Therapeutic azide compounds

ABSTRACT

Pharmaceutical prodrug compositions are provided comprising azide derivatives of drugs which are capable of being converted to the drug in vivo. Azide derivatives of drugs having amine, ketone and hydroxy substituents are converted in vivo to the corresponding drugs, increasing the half-life of the drugs. In addition azide prodrugs are often better able to penetrate the blood-brain barrier than the corresponding drugs. Especially useful are azide derivatives of cordycepin, 2′-F-ara-ddI, AraA, acyclovir, penciclovir and related drugs. Useful azide prodrugs are azide derivatives of therapeutic alicyclic amines, ketones, and hydroxy-substituted compounds, including aralkyl, heterocyclic aralkyl, and cyclic aliphatic compounds, where the amine or oxygen moiety is on the ring, or where the amine or oxygen moiety is on an aliphatic side chain, as well as therapeutic purines and pyrimidines, nucleoside analogs and phosphorylated nucleoside analogs.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of PCT ApplicationPCT/US96/14494 filed Sep. 6, 1996, which takes priority from ProvisionalApplication No. 60/003,383 filed Sep. 7, 1995, incorporated herein byreference to the extent not inconsistent herewith.

GOVERNMENT RIGHTS

[0002] This invention was funded, at least in part, by the U.S.Government under National Institutes of Health Grant No. AI25899.Accordingly, the U.S. Government may have certain rights herein.

FIELD OF THE INVENTION

[0003] This invention is in the field of pharmaceuticals, specificallyazide derivatives of pharmaceutically active compounds.

BACKGROUND OF THE INVENTION

[0004] In recent years the pharmaceutical industry has developed aneffective panoply of therapeutic compounds for the treatment of humandisease. Antibacterial compounds such as penicillin, the sulfa drugs,and more recently, aminoglycocide and cephalosporin antibiotics havedrastically reduced fatalities from bacterial infection. Viralinfections, once thought to be beatable, can now be controlled withantiviral agents, notably nucleoside analogs such as acyclovir andrelated compounds. A diagnosis of cancer, at one time a virtual deathsentence, is now simply a prelude to often-successful treatment withantineoplastic drugs such as methotrexate. Even epilepsy, whose victimswere thought to have been chosen by the gods as special vehicles ofdivine possession, has yielded to the protection of dopantine. AIDSitself, the newest and most frightening of our diseases, has been atleast retarded in its progress by nucleoside replication inhibitors suchas AZT (3′-azido-3′-deoxythymidine).

[0005] Effective as these pharmaceuticals are, however, once inside thepatient's body, many are quickly inactivated by degrading enzymes,particularly deaminases. In some cases, for example when it is necessaryfor the active drug to cross the blood-brain barrier, undesirably largedoses of the drug must be administered in order to ensure enough willremain in circulation long enough to reach the brain in therapeuticquantities. In other cases, drugs must be administered continuously,effectively tying the patient to the iv needle, in order to provideenough active form of the drug in circulation without having toadminister toxically high concentrations.

[0006] It is thus desirable to provide therapeutic compounds in a formwhich will persist for a longer time in the patient's body withoutdegrading than drugs currently in use.

[0007] A number of efforts have been made to improve these effectivepharmaceuticals by increasing their lipophilicity by attachinglipophilic groups such as acetyl or even cholesterol so as to allowfaster penetration into intercellular spaces and compartments withlipophilic barriers, such as the blood-brain barrier. However, thesemeasures have not always been as effective as desired.

[0008] One class of particularly effective antiviral pharmaceuticalswhich has been used in the treatment of herpes viruses as well as otherviruses, particularly in immunocompromised patients such as thoseinfected with the AIDS virus, are nucleoside analogs. These analogs,after phosphorylation by the enzymes of the cell, disrupt DNA synthesisand are thus useful as anticancer agents as well as inhibitors of virusmultiplication. One of the early compounds used for this purpose was5-iodo-2′-deoxyuridine (IDU). [Darby, G. (1995), “In search of theperfect antiviral,” Antiviral Chem. & Chemother. 1:54-63]. This articlediscloses that such drugs also tend to be toxic to normal cells due tothe fact that they inhibit DNA replication. Acyclovir and valaclovir arementioned as particularly useful compounds in this regard because theybecome phosphorylated only within infected cells, and thus inhibit DNAreplication only in these cells. These drugs, however, have low oralbioavailability (15-20%), which limits their usefulness. Again, a methodfor increasing the half-lives of such drugs is needed.

[0009] Vidarabine, 9-(β-D-arabinofuranosyl)adenine (ara-A) wasoriginally discovered as an antitumor agent [Reist, E. J. et al.,“Potential anticancer agents. LXXVI. Synthesis of purine nucleosides ofβ-D-arabinofuranose,” J. Org. Chem. (1962) 27:3274-3279] and in laterstudies, it was shown to be active against herpes simplex virus type 1and 2 [Drach, J. C. and Shipman, C. Jr., “The selective inhibition ofviral DNA synthesis by chemotherapeutic agents: an indicator of clinicalusefulness?” Ann. NY Acad Sci (1977) 284:396-409; Andrei, G. et al.,“Comparative activity of various compounds against clinical strains ofherpes simplex virus,” Eur. J. Clin. Microbiol. Infect. Diseases (1992)11:143-151]. Ara-A is a licensed compound for the treatment of herpessimplex keratitis [Denis, J. et al., “Treatment of superficial herpessimplex hepatitis with Vidarabine (Vira A): A multicenter study of 100cases,” J. Fr. Ophthalmol. (1990) 13:143-150] and encephalitis [Whitley,R. J., “Herpes simplex virus infections of the central nervous system.Encephalitis and neonatal herpes,” Drugs (1991) 42:406-427; Stula, D.and Lyrer, P., “Severe herpes simplex encephalitis: Course 15 yearsfollowing decompressive craniotomy,” Schweiz. Med. Wochenschr. (1992)122:1137-1140; Whitley, R. J., “Neonatal herpes simplex virusinfections,” J. Med. Virol. (1993), Suppl. 1, 13-21]. It has also beenconsidered for the treatment of genital and disseminated herpesinfections [DRUGDEX (R) Information System, Gelman, C. R. and Rumack, B.H., Eds.; MicroMedex, Inc., Englewood, Colo., 84, Expired May 31, 1995],cytomegalovirus encephalitis [Suzuki, Y. et al., “Cytomegalovirusencephalitis in immunologically normal adults,” Rinsho. Shinkeigaku(1990) 30: 168-173], chronic hepatitis B virus (HBV) infection [Chien,R. N. and Liaw, Y. F., “Drug therapy in patients with chronic type Bhepatitis,” J. Formos. Med. Assoc. (1995) 94(suppl. 1):s1-s9; Fu, X. X.,“Therapeutic effect of combined treatment with ara-A, dauricine andChinese herbs in chronic hepatitis B infection,” Chung. Hua. Nei. Ko.Tas. Chih. (1991) 30:498-501] and acute non-lymphoid leukemia[Resegotti, L., “Treatment of acute non-lymphoid leukemia (ANLL) inelderly patients. The GIMEMA experience,” Leukemia (1992) 6 (suppl.2):72-75]. Ara-A may also be an alternative therapy foracyclovir-resistant herpes simplex virus, cytomegalovirus andvaricella-zoster virus infections [Chatis, P. A. and Crumpacker, C. S.,“Resistance of herpes viruses to antiviral drugs,” Antimicrob. AgentsChemother. (1992) 36:1589-1595; Nitta, K. et al., “Sensitivities toother antiviral drugs and thymidine kinase activity ofacyclovir-resistant herpes simplex virus type 1,” Nippon. Ganka. Gakkai.Zasshi (1994) 98:513-519]. However, the use of ara-A as a clinicallyeffective agent is limited due to its rapid deamination to ara-H byadenosine deanmnase (ADA) in vivo [Cass, E. C.,“9-β-D-Arabinofuranosyladenine (Ara-A),” In Antibiotics. Mechanism ofAction of Anti-eukaryotic and Antiviral Compounds; Hahn, F. E., Ed.;Springer-Verlag: New York (1979) V:87-109; Whitley, R. et al.,“Vidarabine: a preliminary review of its pharmacological properties andtherapeutic use,” Drugs (1980) 20:267-282] as well as its poorsolubility in water.

[0010] There were several attempts to prevent the rapid metabolism ofara-A [Plunkett, W. and Cohen, S. S., “Two approaches that increase theactivity of analogs of adenine nucleosides in animal cells,” Cancer Res.(1975) 35:1547-1554], including the co-administration of adenosinedeaminase inhibitors such as deoxycoformycin [Cass, C. E. and Ah-Yeung,T. H., “Enhancement of 9-β-D-arabinofuranosyladenine cytotoxicity tomice leukemia L1210 in vitro by 2′-deoxycoformycin,” Cancer Res. (1976)36:1486-1491; LePage, G. A. et al., “Enhancement of antitumor activityof arabinofuranosyl adenine by 2′-deoxycoformycin,” Cancer Res. (1975)36(4):1481-1485; Cass, C. E. et al., “Antiproliferative effects of 9-β-Darabinofuranosyladenine-5′-monophosphate and related compounds incombination with adenosine deaminase inhibitors against a mouse leukemiaL1210/C2 cells in culture,” Cancer Res. (1979) 39(5):1563-1569;Plunkett, W. et al., “Modulation of9-β-D-arabinofuranosyladenine-5′-triphosphate anddeoxyadenosine-triphosphate in leukemic cells by 2′-deoxycoformycinduring therapy with 9-β-D-arabinfuranosyladenine,” Cancer Res. (1982)42(5):2092-2096; Agarwal, R. P. et al., “Clinical pharmacology of9-β-D-arabinofuranosyladenine in combination with 2′-deoxycoformycin,”Cancer Res. (1982) 42(9):3884-3886] and N⁶-benzoyladenosine [Tritach, G.L. et al., “Synergism between the antiproliferative activities ofarabinofuranosyladenine and N⁶-benzoyladenosine,” Cancer Biochem.Biophys. (1977) 2(2):87-90]. The effects of ara-AMP and ara-A incombination with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) werestudied in mouse leukemia L1210/C2 cell culture and the results werepromising [Cass, C. E. et al., “Antiproliferative effects of 9-β-Darabinofuranosyladenine-5′-monophosphate and related compounds incombination with adenosine deaminase inhibitors against a mouse leukemiaL1210/C2 cells in culture,” Cancer Res. (1979) 39 (5):1563-1569].However, in the clinical trials with the combination of ara-A anddeoxycoformycin some patients developed toxicities [Miser, J. S. et al.,“Lack of significant activity of 2′-deoxycoformycin alone or incombination with adenine arabinoside in relapsed childhood acutelymphoblastic leukemia. A randomized phase II trial from children'scancer study group,” Am. J. Clin. Oncol. (1992) 15:490-493]. Otherapproaches comprise the conjugation of ara-AMP to lactosaminated humanserum albumin [Jansen, R. W. et al., “Coupling of antiviral drug ara-AMPto lactosaminated albumin leads to specific uptake in rat and humanhepatocytes,” Hepatology (1993) 18:146-152] and administration of ara-Ain nanocapsules to improve the pharmacokinetic profiles [Yang, T. Y. etal., “Studies on pharmacokinetics of 9-β-D-arabinosyladeninenanocapsules,” Yao. Hsueh. Hsuech. Pao. (1992) 27:923-927].9-(β-D-Arabinofuranosyl)-6-dimethylaminopurine (ara-DMAP), after itsintravenous administration in rats and monkeys, was rapidly converted to9-(β-D-arabinofuranosyl)-6-methylaminopurine (ara-MAP) and other purinemetabolic end products [Koudriakova, T. et al., “In vitro and in vivoevaluation of 6-azido-2′-3′dideoxy-2′-fluoro-β-D-arabinofuranosylpurine(FAAddP) and6-methyl-2′,3′-dideoxy-2′-fluoro-β-D-arabinofuranosyladenine (FMAddA) asprodrugs of the anti-HIV nucleosides, 2′-F-ara-ddA and 2′-F-ara-ddI,” J.Med. Chem. In Press]. However, less than 4% of the dose of ara-DMAP wasfound to be converted to ara-A and the half-life of ara-A was four timeslonger.

[0011] Recently, in a metabolic study of AZT, Sommadossi et al.[Cretton, E. M. and Sommadossi, J-P, “Reduction of3′-azido-2′,3′-dideoxynucleosides to their 3′-amino metabolite ismediated by cytochrome P450 and NADPH-cytochrome P-450 reductase in ratliver microsomes,” Drug Metab. Dispos. (1993) 21:946-950; Wetze, R. andEclstome, E., “Synthesis and reactions of6-methylsulfonyl-9-β-D-ribofuranosylpurine,” J. Org. Chem. (1975)40(5):658-660] have shown that the azide moiety in AZT is reduced to anamino moiety by the cytochrome-P 450 reductase system.

[0012] Didanosine (ddI) is a synthetic nucleoside analogue structurallyrelated to inosine with proven activity against human immunodeficiencyvirus (HIV) [Faulds, D. and Brogden, R. N., “Didanosine: a review of itsantiviral activity, pharmacokinetics properties and therapeuticpotential in human immunodeficiency virus infection, ” Drugs (1992)44:94-116]. It is approved for use in patients who are intolerant tozidovudine (AZT) or who have deteriorated on zidovudine therapy.However, its various side effects [Tartaglione, T. A. et al.,“Principles and management of the acquired immunodeficiency syndrome.In: Pharmacotherany. A pathophysiologic approach, J. T. DiPiro et al.(Eds.) Appleton and Lange, Norwalk, Conn. (1993) 1837-1867), chemicalinstability in gastric acid and low oral bioavailability of 27-36%(Drusano, G. L. et al., “Impact of bioavailability on determination ofthe maximal tolerated dose of 2′,3′-dideoxyinosine in phase I trials,”Antimicrob. Agents Chemotherapy (1992) 36:1280-1283] limit itsusefulness. Furthermore, there is evidence that ddI enters the centralnervous system and the cerebrospinal fluid (CSF) less readily than doesAZT. The extent of ddI uptake in brain tissue and CSF, relative to thatin plasma, was only 4.7 and 1.5%, respectively [Collins, J. M. et al.,“Pyrimidine dideoxyribonucleosides: selectivity of penetration intocerebrospinal fluid,” J. Pharmacol. Exp. Ther. (1988) 245:466-470;Anderson, B. D. et al., “Uptake kinetics of 2′,3′-dideoxyinosine intobrain and cerebrospinal fluid of rats: intravenous infusion studies,” J.Pharmacol. Exp. Ther. (1990) 253:113-118; Tuntland, T. et al., “Affluxof Zidovudine and 2′,3′-dideoxyinosine out of the cerebrospinal fluidwhen administered alone and in combination to Macaca nemestina,” Pharm.Res. (1994) 11:312-317].

[0013] In an effort to overcome the instability of ddI and2′,3′-dideoxyadenosine (ddA) in acidic conditions, the2′-fluoro-β-D-arabinofuranosyl derivatives 2′-F-ara-ddI and 2′-F-ara-ddAof the nucleosides have been synthesized [Marquez, V. E. et al.,“Acid-stable 2′-fluoro purine dideoxynucleosides as active agentsagainst HIV,” J. Med. Chem. (1990) 33:978-985]. These authors reportedthat 2′-F-ara-ddA and 2′-F-ara-ddI were stable in acidic media and wereas potent as the parent compounds in protecting CD4+ATH8 cells fromcytopathogenic effects of HIV-1. However, 2′-F-ara-ddI, as well as ddI,are relatively hydrophilic and do not readily penetrate the blood-brainbarrier (BBB) in mice [Shanmuganathan, K. et al., “Enhanced braindelivery of an anti-HIV nucleoside 2′-F-ara-ddI by xanthine oxidasemediated biotransformation,” J. Med. Chem. (1994) 37:821-827]. Recently,applicants synthesized a more lipophilic prodrug,2′,3′-dideoxy-2-fluoro-β-D-arabinofuranosyl-purine (2′-F-ara-ddP), whichwas converted to the parent nucleoside, 2′-F-ara-ddI by xanthine oxidasein vivo. Pharmacokinetic studies indicated 2′-F-ara-ddP increased thedelivery of 2′-F-ara-ddI to the brain in mice. TheAUC_(brain)/AUC_(serum) ratio for 2′-F-ara-ddI was increased toapproximately 36% after oral and intravenous prodrug administration[Shanmuganathan, K. et al., “Enhanced brain delivery of an anti-HIVnucleoside 2′-F-ara-ddI by xanthine oxidase mediated biotransformation,”J. Med. Chem. (1994) 37:821-827].

[0014] Cordycepin is potentially very active against tumor growth andviral replication [Yeners, K. et al., “Cordycepin selectively killsTdT-positive cells,” Abstract of presentation to American Soc. of Clin.Oncology Meeting, May 1993]. However, the effectiveness of cordycepin invivo is markedly decreased by rapid deamination. Cordycepin exhibits itsbiological activity through direct inhibition of viral replicationthrough its ability to block polyadenylic acid [poly(A)] synthesis, thusinterfering with processing and maturation of both cellular and viralmRNA. [Svendsen, K. R. et al. (1992), “Toxicity and metabolism of3′-deoxyadenosine N¹-oxide in mice and Ehrlich ascites tumor cells,”Cancer Chemother. Pharmacol. (1992) 30:86-94.] Cordycepin isphosphorylated by adenosine kinase to 3′-deoxyadenosine monophosphate,with further phosphorylation by adenylate kinase to 3′-deoxyadenosinetriphosphate which exerts toxic effects due to its incorporation intoRNA in lieu of ATP, thereby functioning as a chain terminator.

[0015] In vivo, however, the effectiveness of cordycepin as an antitumoragent is limited because of very rapid deamination of the compound toyield 3′-deoxyinosine which is biologically inert. That reaction iscatalyzed by adenosine deaminase. [Frederiksen, S. and Klenow, H.(1975), “3-Deoxyadenosine and other polynucleotide chain terminators,”In Handbook of experimental pharmacology, (A. C. Sartorelli and G.Johnes, Eds.) 657-669.]

[0016] The in vivo antitumor activity of cordycepin can be enhanced byadministration with adenosine deaminase inhibitor 2′-deoxycoformycin(2′-DCF). Administered together, cordycepin and 2′-DCF resulted inmarked inhibition of L1210 and P388 cell growth in vitro and in micemodels in vivo. [Johns, D. G. and Adamson, R. H. (1976), “Enhancement ofthe biological activity of cordycepin (3′-deoxyadenosine) by theadenosine deaminase inhibitor 2′-deoxycoformycin,” Biochem. Pharmacol.(1976) 25:1441-1444.]

[0017] Another way to avoid deamination of cordycepin is through the useof 3′-deoxyadenosine N¹-oxide (3′-dANO) as a prodrug. 3′-dANO ismetabolically inert until it has entered a target cell that is capableof reducing 3′-dANO to cordycepin. [Svendsen, K. R. et al. (1992),“Toxicity and metabolism of 3′-deoxyadenosine N¹-oxide in mice andEhrlich ascites tumor cells,” Cancer Chemother. Pharmacol. (1992)30:86-94.]

[0018] Reduction to the amine has been observed to adversely affect thebioavailability of other drugs as well. Reduction to the amine has beenshown to deactivate the antitumor agent meta-azidepyrimethamine.[Kamali, F., et al. (1988), “Medicinal azides. Part 3. The metabolism ofthe investigational antitumor agent meta-azidepyrimethamine in mousetissue in vitro,” Xenobiotica 18:1157-1164.]

[0019] This invention provides azide compounds, preferably azidederivatives of therapeutically active substances which provide increasedhalf-lives for the therapeutically active substances.

[0020] Azide derivatives of certain biologically active compounds havebeen synthesized for the purpose of optical imaging. [Nicholls, D., etal. (1991), “Medicinal azides. Part 8. The in vitro metabolism ofp-substituted phenyl azides,” Xenobiotica 21:935-943.]

[0021] Azide drugs such as 3′-azido-3′-deoxythymidine (AZT, also knownas zidovudine) have been used in the treatment of AIDS because of theirinhibition of viral replication. [Tartaglione, T. A., et al. (1993),“Principles and management of the acquired immunodeficiency syndrome.In: Pharmacotherapy. A pathophysiologic approach, DiPiro, J. T. et al.,eds., Appleton and Lange, Norwalk, Colo. 1837-1867.] AZT is reduced invivo to the corresponding amino compound. [Placidi, L., et al. (1993,“Reduction of 3′-azido-3′-deoxythymidine to 3′-amino-3′-deoxythymidinein human liver microsomes and its relationship to cytochrome P450,”Clin. Phannacol. Ther. 54:168-176. This is also true ofazidodideoxynucleosides. Cretton, E. M. and Sommadossi, J-P (1993),“Reduction of 2′-azido-2′,3′-dideoxynucleosides to their 3′aminometabolite is mediated by cytochrome P-450 and NADPH-cytochrome P450reductase in rat liver microsomes,” Drug Metab. Dispos. 21:946-950.] Thedegradation product of AZT is not therapeutically effective and is, infact, toxic. [Cretton, E. M. et al., “Catabolism of3′-azide-3′-deoxythymidine in hepatocytes and liver microsomes withevidence of formation of 3′-amino-3′-deoxythimidine, a highly toxiccatabolite for human bone marrow cells,” Molec. Pharmacol. (1991)39:258-266.]

[0022] Kumar, R., et al. (1994), “Synthesis, in vitro biologicalstability, and anti-HIV activity of 5-halo-6-alkoxy (or azide)-5,6-dihydro-3′-azido-3′deoxythymidine (AZT),” J. Med. Chem.37:4297-4306, reported that 6-azide derivatives of AZT were 2-3 logunits less active than AZT.

[0023] An azide derivative of 2,6-diaminopurine as well as several otherderivatives of 2,6-diaminopurine have also been recognized as potentinhibitors of HIV replication. These compounds also inhibit adenosinedeaminase and inhibit the deamination of9-beta-D-arabinofuranosyladenine (araA). [Balzarini, J. and DeClercq, E.(1989), “The antiviral activity of 9-beta-D-arabinofuranosyladenine isenhanced by the 2′,3′-dideoxyriboside, the2′,3′-didehydro-2′,3′-dideoxyriboside and the3′-azido-2′,3′-dideoxyriboside of 2,6-diaminopurine,” Biochem. Biophys.Res. Commun. 159:61-67.]

[0024] A method as provided herein, is needed for increasing thehalf-life of pharmaceutically active compounds so as to avoid problemsassociated with the rapid degradation of the compounds in the patient'sbody.

SUMMARY OF THE INVENTION

[0025] This invention provides pharmaceutical compositions whichcomprise azide compounds, preferably azide derivatives of biologicallyactive therapeutic compounds (also referred to herein as “drugs”). Theazide derivatives of this invention are reduced in vivo to thecorresponding drug.

[0026] Many therapeutic compounds are quite effective, but do notpersist in the system as long as desired to produce the best effect.Drugs comprising amine moieties are susceptible to deamination in thebody, which often destroys their effectiveness. When the amine moietiesof these drugs are converted to azide (N₃) moieties, the azides arereduced to the corresponding amines, thus converting the azidederivatives to the active amine drugs, such that the active forms of thedrug have a longer half life, continuing to exhibit biological activityin the in vivo system for a longer period of time after administrationthan the drugs themselves. Drugs which are ketones or have hydroxysubstituents may also advantageously be converted to the correspondingazides to increase their half-lives.

[0027] In addition to having increased half-lives, azide derivativesoften are better able to penetrate the cells and compartments withlipophilic barriers, such as the prostate capsule and blood-brainbarrier, so that they are more effective in reaching the site wheretheir activity is desired than the corresponding amine, ketone orhydroxy form of the drug. The azide derivatives of therapeuticallyactive substances of this invention are sometimes referred to herein as“prodrugs” for the drugs of which they are derivatives.

[0028] As an example of this invention, the azide derivative ofcordycepin, an active anticancer agent, has been prepared.N⁶-azido-β-D-3′-deoxyribofuranosyl purine (ADRP), having an azide groupin place of the amine group of cordycepin, is reduced in vivo tocordycepin, an active anticancer agent. Thereafter, the cordycepin isfurther deaminated to the inactive 3′-deoxyinosine metabolite:

[0029] In addition, drugs which in their active form have a carbonylmoiety may be converted to the corresponding azides. The body reducesthese to the corresponding amines, then further reduces them to theactive carbonyl form by deaminases in vivo. For example, thecorresponding azide of 2′-fluoro-2′,3′-dideoxyinosine (2′-F-ara-ddI),namely 6-azido-2′,3′-dideoxy-2′-fluoro-β-D-arabinofuranosylpurine(FAAddP), has been prepared. When this azide derivative is introducedinto the body, it is first reduced to the corresponding amine,2′-fluoro-2′,3′-dideoxyadenosine (2′-F-ara-ddA) which is inactive, thendeaminated to the active carbonyl form, 2′-F-ara-ddI:

[0030] Drugs having a hydroxy form may similarly be administered asazide derivatives which are reduced in vivo first to the ammne, then thehydroxy form. For example, (−)β-D-2,6-amino-azidopurine dioxolane (DAPD)is reduced to the diamino derivative which is then deaminated to thecorresponding hydroxy form, (−)-β-D-Dioxolane guanine (DXG):

[0031] Preferably, the drugs converted to azides herein are drugs havingprimary amine ring substituents, which are capable of being reduced fromthe azide to the corresponding amine. Other preferred drugs arealiphatic amines, drugs having secondary or tertiary amine ring or chainsubstituents, drugs with oxygen substituents attached to carbon atoms(i.e. ketones) or hydroxy substituents attached to carbon atoms, whichare capable of being reduced from the azide to the corresponding amine,and from the amine to the ketone or hydroxy form.

[0032] Accordingly, this invention provides a pharmaceutical compositioncomprising:

[0033] a) an azide derivative of a drug, said azide derivative beingcapable of being converted to the drug in vivo;

[0034] b) a suitable pharmaceutical carrier.

[0035] Azides which are capable of being converted to the correspondingamines in vivo include azides corresponding to aryl, ring-substitutedaryl, and aliphatic amines, preferably primary amines, and azidescorresponding to drugs having carbonyl or hydroxy groups, such as oxygensubstituted aryl and aliphatic compounds.

[0036] These azides are selected from the group consisting of azidederivatives of biologically active purines and pyrirnidines, nucleosideanalogs, phosphorylated nucleoside analogs, aminoglycoside antibiotics,ampicillin and ampicillin analogs, sulfonamides, cephalosporin andcephalosporin analogs, and other alicyclic amines, ketones, and hydroxycompounds, including aralkyl, heterocyclic aralkyl, and cyclic aliphaticcompounds, where the amine or oxygen moiety is on the ring, or where theamine or oxygen moiety is on an aliphatic side chain.

[0037] The most preferred compounds of this invention are2′-F-azido-ara-ddP, an azide derivative of 2′-F-ara-ddI;9-(β-D-arabinofaranosyl)-6-azidopurine (6-AAP) an azide derivative ofadenine arabinoside (AraA); and N⁶-azido-β-D-3′-deoxyribofuranosylpurine, an azide derivative of cordycepin.

[0038] This invention also provides a method of increasing the half-lifeof a drug in a subject comprising administering to the subject an azidederivative of said drug capable of being reduced to the drug in thepatient's body. Following administration of the azide derivative, thepatient's serum levels may be monitored to determine the presence of thedrug, or specific effects of the drug may be monitored.

[0039] The methods of this invention also include a method forameliorating a pathological condition in a patient comprising treatingthe patient with a therapeutically effective azide compound, preferablyone which is capable of metabolizing in vivo to a therapeutic compoundeffective for the treatment of said pathological condition. This methodalso includes co-administering said azide compound with othertherapeutic agents.

[0040] Azides are converted by reductase in the subject's body to thecorresponding amines, and are then further deaminated. There are anumber of deaminases in the body, including adenosine deaminase andcytidine deaminase, which are capable of further metabolizing amines tothe corresponding ketones or hydroxy-substituted compounds. The activedrug may be the amine, or the ketone or hydroxy compound.

BRIEF DESCRIPTION OF THE FIGURES

[0041]FIG. 1 shows pharmacokinetic profiles of the azide derivative ofcordycepin, N⁶-azido-β-D-3′-deoxyribofuranosyl purine, cordycepin andmetabolite after oral () and intravenous ({overscore (V)})administration of the azide derivative to mice at a dose of 100 mg/kg.FIG. 1A shows the profile for the azide derivative, FIG. 1B shows theprofile for cordycepin, and FIG. 1C shows the profile for the3′-deoxyinosine metabolite.

[0042]FIG. 2 shows mean ±SD serum () and brain (∘) concentrations of2′-F-ara-ddI after intravenous administration of 20 mg/kg 2′-F-ara-ddIto mice.

[0043]FIG. 3 shows mean ±SD concentrations of FAAddP (∘), 2′-F-ara-ddA({overscore (V)}) and 2′-F-ara-ddI () in serum (A) and brain (B) afterintravenous administration and in serum (C) after oral administration of55 mg/kg FAAddP to mice.

[0044]FIG. 4 shows mean ±SD serum (A) and brain (B) concentrations ofFMAdda () and 2′-F-ara-ddI ({overscore (V)}) after intravenousadministration of 55 mg/kg FMAddA to mice.

[0045]FIG. 5 shows mean ±SD concentrations of 6-AAP in serum (μg/ml)after intravenous ({overscore (V)}) and oral (▾) administration, and inbrain (μg/g) after intravenous (∘) and oral () administration of 100mg/kg of 6-AAP to mice.

[0046]FIG. 6 shows mean ±SD serum concentrations of ara-A afterintravenous administration of ara-A (▾) and ara-A after oral () andintravenous ({overscore (V)}) administration of 100 mg/kg of 6-AAP tomice.

[0047]FIG. 7 shows mean ±SD brain concentrations of ara-A after oral({overscore (V)}) and intravenous ({overscore (V)}) administration of100 mg/kg of 6-AAP to mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] A preferred class of the azide derivatives of this invention areazide derivatives of biologically active purines and pyrimidines,nucleoside analogs, and phosphorylated nucleoside analogs. For example,azide derivatives are prepared from the following drugs: dideoxyinosine(ddI), for which the azide derivative is6-azido-(2′,3′-dideoxy-β-D-glycero-pentofuranosyl) purine; 2′-F-ara-ddI,for which the corresponding azide derivative is6-azido-2′,3′-dideoxy-2′-fluoro-β-D-arabinofuranosylpurine (FAAddP);arabinofuranosyladenine (araA), for which the corresponding azidederivative is 9-(β-D-arabinofuranosyl)-6-azidopurine (6-AAP);cordycepin, for which the corresponding azide derivative isN⁶⁻azido-β-D-3′-deoxyribofuranosyl purine; flucytosine for which thecorresponding azide derivative is 4-azido-5-fluoro-2(1H)-pyrimidinone;cytarabine, for which the corresponding azide derivative is4-azido-1-β-D-arabinofuranosyl-2(1H)-pyrimidinone;1-(fluoroarabino)fluorocytosine, for which the corresponding azidederivative is4-azido-5-fluoro-1-(β-D-arabinofuranosyl-2(1H)-pyrimidinone;trimethoprim, for which the corresponding azide derivative is2,4-diazido-5-(3,4,5-trimnethoxybenzyl)pyramidine;1-(fluoroarabino)fluorocytosine, for which the corresponding azidederivative is4-azido-5-fluoro-1-β-D-arabinofuranosyl-2(1H)-pyrimidinone;arabinocytidine, for which the corresponding azide derivative is4-azido-1-β-D-ribofuranosyl-2-(1H-pyrirndinone; and2-chloro-9-arabinodeoxyadenosine, for which the corresponding azidederivative is 2-chloro-6-azido-5-arabinopurine; and purine-derivedantibiotics such as acyclovir, for which the corresponding azidederivative is2-amino-6-azido-1,9-dihydro-9[(2-hydroxyethoxy)methyl]-purine;penciclovir, for which the corresponding azide derivative is2-amino-6-azido-1,9-dihydro-9-[dihydroxymethyl]propyl-purine; andganciclovir for which the corresponding azide derivative is2-amino-6-azido-1,9-dihydro-9-[dihydroxymethylmethoxymethyl]-purine.

[0049] A preferred class of nucleoside analogs are the nucleosideanalogs used as antivirals, for example in the treatment of herpessimplex virus (HSV), including the pyrimidine nucleoside analogsidoxuridine (IDU), trifluorothymidine (F₃T), and the purine nucleosideanalogs vidarabine, acyclovir, valaciclovir, ganciclovir, penciclovir,and famciclovir.

[0050] A further preferred class of nucleoside analogs includes theforegoing along with β-D-5-Fluoro-1′,3′-dioxalane cytosine (β-D-FDOC),β-D-5-Fluoro-2′,3′-dideoxycytidine (β-D-FddC),β-D-5-Fluoro-2′,3′-dideoxy-2′,3′-didehydrocytosine (β-D-Fd4C),β-D-5-Fluoro-3′deoxy-3′thiacytidine (β-D-FTC), adenosine,2′-deoxyadenosine, cytidine, 2′-deoxycytidine, 5′-fluorocytosine, andarabinofuranosylcytosine (ara-C), all of which are converted to thecorresponding azide by treatment with the appropriate azide donor, e.g.N₃.

[0051] Monophosphates, diphosphates and triphosphates of the azidederivates of nucleoside analogs also constitute a preferred class ofcompounds of this invention.

[0052] Another preferred class of azide derivatives are azidederivatives of aminoglycoside antibiotics which are primary amines,ketones, or hydroxy-substituted compounds, such as gentamycin,tobramycin and kanamycin.

[0053] A further preferred class of azide derivatives are azidederivatives of ampicillin and its analogs such as bacampicillin, forwhich the corresponding azide derivatives are 6-[(azidephenylacetyl)amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo-[3.2.0]heptane-2-carboxylicacid and ester, respectively.

[0054] A further preferred class of azide derivatives are azidederivatives of sulfonamides which are primary amines or ketones, such asp-azidebenzenesulfonamide and its analogs. Azide derivatives can besynthesized by means known to the art corresponding to the followingsulfa drugs: sulfabenz, sulfabenzamide, sulfabromomethazine,sulfacetamide, sulbenox, sulfacytine, sulfadiazine, sulfadicramide,sulfadimethoxine, sulfadoxine, sulfaethidole, sulfaaguanidine,sulfaguanole, sulfalene, sulfamerazine, sulfameter, sulfamethazine,sulfamethomidine, sulfamethoxazole, sulfamethoxypyridazine,sulfametrole, sulfamidochrysoidine, sulfamoxole, sulfanilamide,sulfanilamidomethanesulfonic acid triethanolate, sulfanilamidosalicylicacid, sulfanilic acid, 2-p-sulfanilyanilinoethanol,p-sulfanilylbenzylamine, sulfanilyl urea, N-sulfanilyl-3,4-xylamide,sulfanitran, sulfaperine, sulfaphenazole, sulfaproxyline, sylfapyridine,sulfaquinoxaline, sulfasomizole, sulfasymazine, sulfathiazole,sulfaathiourea, sulfazamet, 4,4′-sulfinyldianiline, sulfisomidine andsulfisoxazole.

[0055] A further preferred class of azide derivatives are azidephenylderivatives of cephalosporin or its biologically active analogs whichare primary amines or ketones, such as cephalexin, for which thecorresponding azide derivative is[7-[azidephenylacetyl)amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid; cephaloglycin, for which the corresponding azide derivative is3-[(acetyloxy)methyl)-7-[(azidephenylacetyl)amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid; cephalosporin C, for which the corresponding azide derivative is7-(D-5-azido-5-carboxyvaleramido)-3-(carboxy)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxyliacid;cephamycins for which the corresponding azide derivatives are7-(D-5-azido-5-carboxyvaleramido)-7-methyloxy-3-(carboxy)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid esters, and cephadrine.

[0056] A further preferred class of azide derivatives are azidederivatives of biologically active alicyclic amines, ketones, andhydroxy-substituted compounds, including aralkyl, heterocyclic aralkyl,and cyclic aliphatic compounds, where the amine or oxygen moiety is onthe ring, e.g. trimetrexate, for which the corresponding azidederivative is2,4-diazido-5-methyl-6-[3,4,5-trimethoxyanilino)methyl]quinazoline;procaine, for which the corresponding azide derivative isp-azidebenzoyldiethylaminoethanol; dapsone, for which the correspondingazide derivative is 4,4′-diazidodiphenyl sulfone; amantadine, for whichthe corresponding azide derivative is1-azidetricyclo[3.3.1.1^(3.7)]decane; and methotrexate, for which thecorresponding azide derivative isN-[4-[[(2,4-Diazido-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid; or where the amine or oxygen moiety is on an aliphatic side chain,e.g. amphetamine, for which the corresponding azide derivative is1-phenyl-2-azidepropane; L-dopa, for which the corresponding azidederivative is 2-azido-3-(3,4-dihydroxyphenyl)propanoic acid;trimethoprim, for which the corresponding azide derivative is2,4-diazido-5-(3,4,5-trimethoxybenzyl)pyrimidine; and histamine, forwhich the corresponding azide derivative is β-azidoethylimidazole.

[0057] A further preferred class of azide derivatives are azidederivatives of biogenetic amines including epinephrin, norepinephrin,dopamine, and seratonin.

[0058] Corresponding azides may be formed for drugs useful for virtuallyany therapeutic purpose, so as to increase the half-lives of said drugs.For example, in addition to drugs which are antibacterial, antiviral,antifungal, local anesthetic and cancer therapeutic, for which exampleshave been given above, corresponding azides may be made for diuretics,for example, furosemide; anesthetics, for example ketamine;non-steroidal anti-inflammatories, for example 3-amino-4-hydroxybutyricacid; psychiatric drugs, for example Prozac; beta-blockers, for examplepropranolol; hormones, for example thyroid; and others. Suitable drugsmay be identified by those skilled in the art by reviewing compilationsof therapeutic compounds such as Merck's Manual, and identifying thosehaving amine, carbonyl or hydroxy substituents. Formulation ofcorresponding azides may be readily accomplished by those of ordinaryskill in the art without undue experimentation by means known in the artsuch as are described herein.

[0059] As will be appreciated by those skilled in the art, compoundshaving more than one amino, carbonyl, or hydroxy moiety may have all, atless than all, e.g. only one, of these substituents converted to azidegroups.

[0060] The azide derivatives of therapeutic compounds claimed herein bymeans of their chemical names include the free forms as well aspharmaceutically acceptable salts (including esters) thereof. Dependingon the physico-chemical characteristics of the drug, saidpharmaceutically acceptable salt may further be either inorganic acidaddition salts such as hydrochloride, hydrobromide or sulfate; organicacid addition salts such as citrate, acetate, oxalate, hibenzate,methanesulfonate, alkali metal salts such as sodium salt or potassiumsalt, alkaline earth metal salts such as calcium salt or magnesium salt,amine salts, and the like.

[0061] The dose of the azide derivative to be used is not critical, butthe azide derivative is preferably used within the usual dosage range ofthe drug to which it reduces. As will be appreciated by those skilled inthe art, dosage amount and timing may advantageously be varied in lightof the longer half-lives of the drugs when the azide derivatives areadministered, as compared to the case in which the drug itself isadministered.

[0062] The azide derivatives of therapeutic compounds hereof may beadministered to a subject or patient which is a warm-blooded animal,including man, via any means known to the art, including intravenously,by injection, and through conventional enteral routes, for example,orally or rectally. Further these compounds may be administered in theform of conventional preparations, for example, in solid dosage formssuch as tablets, pills, powders, granules or suppositories; or in liquiddosage forms such as solutions, syrups, emulsions, elixirs, suspensionor lemonades. In formulating these preparations, there may be used apharmaceutically acceptable carrier such as a binder e.g., syrup, arabicgum, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, a diluent suchas lactose, sucrose, corn starch, calcium phosphate, sorbit and thelike, a lubricant such as magnesium stearate, talc, polyethylene glycol,silica, and the like, a disintegrator, such as corn starch, a wettingagent such as sodium lauryl sulfate, a suppository base such as cacaobutter, laurin butter, polyethylene glycol, eg. macrogol, glycerinatedgelatin, triglyceride of saturated fatty acids C12-D18, a flavoringagent, sweetening agent, or coloring agent. The term “pharmaceuticallyacceptable carrier” as used herein does not include water alone oralcohols alone, although any of the foregoing additives in combinationwith water may constitute a pharmaceutically acceptable carrier.

[0063] In general, azide derivatives may be prepared by means known tothe art. Azide derivatives of aromatic ketones, hydroxy-substitutedcompounds or amines may be synthesized starting with the correspondingchloride and exchanging with sodium or lithium azide. For example,azidocytosine analogs may be prepared from uracil analogs according tothe following scheme:

[0064] Azidoadenine analogs may be prepared from guanine analogsaccording to the following scheme:

[0065] The synthesis of the azide derivative for acyclovir,2-amino-6-azido-1,9-dihydro-9[(2-hydroxyethoxy)methyl]-purine, is shownin the following scheme:

[0066] The synthesis of the azide derivative for penciclovir,2-amino-6-azido-1,9-dihydro-9-[dihydroxymethyl]propyl-purine is shown inthe following scheme: 22

[0067] The synthesis of the azide derivative for ganciclovir,2-amino-6-azido-1,9-dihydro-9[dihydroxymethylmethoxymethyl]-purine, isshown in the following scheme:

[0068] The synthesis of the azide derivative for cytarabine,4-azido-1-β-D-arabinofuranosyl-2(1H)-pyrimidinone, is shown in thefollowing scheme:

[0069] The synthesis of the azide derivative for2-chloro-9-arabinodeoxyadenosine (2-CdA),2-chloro-6-azido-5-arabinopurine, is shown in the following scheme:

[0070] Aliphatic compounds may be prepared by treating withtrifluoromethylsulfonyl azide according to the following scheme:

[0071] Care must be taken in the synthesis of aliphatic azidederivatives due to the explosive nature of trifluoromethylsulfoyl azide.As will be appreciated by those skilled in the art, care must also betaken to preserve the stereochemistry of the drug being converted to thecorresponding azide.

EXAMPLES Example 1

[0072] Synthesis of Cordycepin Derivative.

[0073] The cordycepin derivative, N⁶-azido-β-D-3′-deoxyribofuranosylpurine, was synthesized according to the following scheme:

[0074] Diaceton-D-glucose (156 g 0.6 mol.) was stirred in 1000 ml CHCl₂.To this solution were added pyridimium dichromate (PDC 135 g 0.36 mol)and Ac₂O (186 ml 1.98 mol). The mixture was refluxed for 30 min. Afterconcentration, the residue was diluted with EtOAc (500 ml), filtered andthe filtrate was filtered through a silica gel pad and was washed withEtoAc. The combined filtrate was concentrated and was evaporated withtoluene to give an oil, 110 g, yield: 70.8%.

[0075] A solution of this oil (110 g 0.42 mol) and TSNHNH₂ (86.1 g, 0.46mol) in absolute EtOH (600 ml) was refluxed for 3 hours. After cooling,the white crystalline product was filtered and washed with MeOH (60 g.,yield: 39%).

[0076] To a stirred solution of this white crystalline substance (60 g,0.23 mol) in THP-MeOH (1:1, 700 ml) was added a trace of methyl orangeand NaCNBH₃ (15.1 g, 10.24 mol). Methanolic HCl was added dropwisekeeping the color of the solution at the red-yellow transition point.The mixture was stirred at room temperature for one hour. A secondportion of NaCNBH₃ (8.7 g, 0.14 mol) was added, followed by dropwiseaddition of methanolic HCl to maintain pH 3. After stirring at roomtemperature for one hour, the mixture was neutralized with NaHCO₃ andwas concentrated to dryness. The residue was dissolved in H₂O (150 ml)and was extracted with CH₂Cl₂ (200 ml×3), the organic layer was washedwith brine and dried, filtered and evaporated to give a yellowish oilwhich was purified by silica gel column to give compound A as an oil (30g) and compound B as a white solid (30 g). Compound A was dissolved in60% HOAc (450 ml) and was stirred at room temperature overnight. Themixture was neutralized with NaHCO₃ (solid) and was extracted withCH₂Cl₂ (200 ml×3). The organic layer was washed with brine, dried, andfiltered and evaporated to give a yellowish oil which was purified bysilica gel column to give another 16 g compound B. Total yield: 84.5%.

[0077] A mixture of Compound B (46 g, 0.118 mol) and NaOAc.3H₂O (64.6 g,0.48 mol) in absolute EtOH (600 ml) was refluxed for one hour. Themixture was concentrated to dryness, the residue was dissolved in H₂O(150 ml) and was extracted with EtOAc (200 ml×5). The organic layer wascombined and was washed with brine (200 ml×2), dried and filtered, thenevaporated to give a yellowish oil which was purified by silica gelcolumn to give a yellowish oil. 20 g, yield: 83%.

[0078] To a solution of this yellowish oil (20 g, 0.099 mol) in MeOH(300 ml), NaIO₄ (24.6 g, 0.115 mol) in H₂O (200 ml) was added at 0° C.After the mixture was stirred for 10 minutes, NaBH₄ (5.7 g, 0.149 mol)was added and the mixture was stirred for another ten minutes. Afterfiltration, the filtrate was concentrated to dryness and the residue waspurified by silica gel column to give a white solid. 14 g, yield: 82.8%.

[0079] A mixture of this white solid (3.45 g, 20 mmol), DMAP (50 mg)Ac₂O (2.26 ml, 24 mmol) and Et₃N (4.18 ml. 30 mmol) in CH₂Cl₂ (50 ml)was refluxed for one hour. After cooling, the mixture was washed withwater and brine, the organic layer was dried, filtered and evaporated todryness to obtain a yellowish oil. 4.1 g, yield: 95%.

[0080] A mixture of this yellowish oil (13.5 g, 62.9 mmol) in 50 ml 80%HOAc was stirred at 70° C. for 24 hours. The mixture was refluxed foranother hour. The mixture was concentrated to dryness and wascoevaporated with toluene. The brown residue was dissolved in CH₂Cl₂(200 ml) and Ac₂O (6.5 ml, 69.2 mmol), Et₃N (11.4 ml, 81.8 mmol),DM)0.AP (100 mg) were added. The mixture was refluxed for one hour. Dueto the incompletion of the reaction, another 2 ml Ac₂O, 4 ml Et₃N wereadded, and the mixture was refluxed for another three hours. Aftercooling, the mixture was washed with H₂O (50 ml×2), dried, filtered andconcentrated to dryness. The residue was purified by silica gel columnto give a light yellow oil.

[0081] A mixture of 6-chloropurine (0.45 g, 2.9 mmol), HMDS (12 ml) andammonium sulfate (50 mg) was refluxed for one hour. The resulting clearsolution was concentrated in vacuo under anhydrous conditions. Theresidue was dissolved in dry Ch₂Cl₂ (75 ml) and was cooled to 0° C. Tothis cooled solution, the light yellow oil (0.5 g, 1.9 mmol) in CH₂Cl₂(10 ml) and TMSOH (fresh opened, 0.6 ml, 3 mmol) were added. Thetemperature was then brought up to room temperature and the mixture wasstifrred for 30 minutes. The mixture was stirred at room temperature foranother hour. Saturated NaHCO₃ was added to quench the reaction. Afterseparation, the aqueous layer was extracted with EtOAc (50 ml×2). Thecombined organic layer was dried, filtered and evaporated to dryness.The residue was then purified by silica gel column to obtain a colorlessoil. 450 mg, yield: 66.1%.

[0082] To a solution of this colorless oil (3.69 g, 10.4 mmol) in MeOH(300 ml) was added saturated Na2CO₃ (3 ml to pH=8). The mixture wasstirred at room temperature for 30 minutes. The mixture was neutralizedwith HOAc to pH 6 to 7. The mixture was concentrated to dryness and wascoevaporated with toluene to give a white solid. To this solid, DMF (700ml) and NaN₃ (1.1 g, 17 mmol) were added and the mixture was stirred at60° C. for three hours. After concentration, the residue was melted withHOAc (100 ml). The white solid was filtered out, the filtrate wasconcentrated to dryness. After adding MeOH (50 ml), a white solid wasprecipitated. After filtration, a white crystal was obtained. 1.4 g,yield: 48.6%.

Example 2

[0083] Bioactivity of Cordycepin Derivative.

[0084] Female NIH Swiss mice (Harland Sprague-Dawley, Indianapolis,Ind.) weighing 24-28 g were used for pharmacokinetic experiments. 100mg/kg of cordycepin or the azide derivative of cordycepin wereadministered intravenously (iv) (dissolved in methylsulfoxide (20mg/ml)), or orally (dissolved in 30% glycerin (8.3 mg/ml)). Threeanimals for each time point were sacrificed at 0.08, 0.25, 0.5, 0.75,1.0, 1.5, 2.0, 3.0 and 4.0 h after drug administration. Blood (serum)was collected and immediately analyzed.

[0085] Concentrations of the inosine derivative, cordycepin and theazide derivative of cordycepin in serum were measured byhigh-performance liquid chromatography (HPLC).

[0086] To measure cordycepin and its metabolite concentrations in serum,200 pl serum sample, 50 μl of internal standard 2′-F-ddI (5 μg/ml) and50 μl 2 M perchloric acid as a protein precipitant were added topolypropylene microcentrifuge tubes (1.5 ml). An AzdU was used as aninternal standard (20 μg/ml) to measure the azide derivativeconcentrations in serum. Tubes were vortexed and centrifuged at 9,000rpm for 5 min.

[0087] To neutralize, perchloric acid supernatant was transferred into aclean tube with 160 μl of concentrated sodium tetraborate solution (pH6.5). Tubes were vortexed again and centrifuged at 12,000 rpm for 15min. Samples were held at 0° C. during analysis.

[0088] Chromatographic separations were carried out on a Shimadzygradient HPLC system (Shimadzy Corporation, Kyoto, Japan), which wasequipped with a Model SPD-10A UV detector, a Model L-10AS pump, a ModelSIL-10A autosampler, a controller Model SCL-10A and a Model CR-501reporting integrator. Chromatography was performed on an AlltechHypersil ODS (5 μm particle size, 4.6×250 mm, Ailtech Associates,Deerfield, Ill.) to determine cordycepin and its metabolite. The azidederivative of cordycepin was analyzed on an Alltech Hypersil BDS column(5 μm particle size, 4.6×250 mm, Alltech Associates, Deerfield, Ill.).

[0089] The mobile phase for cordycepin and metabolite analysis in serumconsisted of 3% acetonitrile in 20 mM sodium borate and 10 mM EDTA (pH6.5) at a flow of 1.4 ml/min and the mobile phase for azide derivativeanalysis in serum consisted of 3 % acetonitrile 20 mM in potassiumphosphate (pH 4.0) at a flow of 1.5 ml/min. The UV detector was set at258 nm for analysis of cordycepin and metabolite and at 290 nm for theazide derivative.

[0090] Standard curves were prepared for each type of sample by addingknown amounts of compounds to serum and subjecting them to theextraction procedure as described above. The limit of quantitation was0.05 μg/ml for cordycepin and metabolite and 0.5 μg/ml for the azidederivative.

[0091] Plasma concentration versus time data for nucleosides wereanalyzed by noncompartment methods. The area under concentration versustime curves (AUC) from time zero to the last measured concentration wasdetermined by the linear trapezoidal rule and the AUC from the time ofthe last measured concentration to infinity was determined by dividingthe last determined concentration by the least squares elimination rateconstant (λ_(Z)). Half-life was calculated from 0.693/λ_(Z). Thevariance of estimated AUC values was calculated as described by Rocci,M. L. Jr. and Jusko, W. J., “LAGRAN program for area and moments inpharmacokinetic analysis,” Comp. Prog. Biomed. (1983) 16:203-216.

[0092] Absolute bioavailability (F) of the azide derivative wascalculated from AUC_(or)/AUC_(iv), where AUC values were determined fromserum nucleoside concentration versus time data. Relative exposures forcordycepin and its metabolite in serum after or versus iv administrationof the derivative were calculated from AUC_(or)/AUC_(iv). TABLE 1Pharmacokinetic parameters of azide derivative, cordycepin andmetabolite in mouse serum after intravenous and oral administration ofazide derivative at a dose of 100 mg/kg AUC_(0→∞)(mg h/L) T_(½), (hr)Compound iv oral F r_(e) iv oral Azide Deriv. 47.2 ± 4.01 44.5 ± 2.78*0.94 — 0.36 0.53 Cordycepin 2.17 ± 0.35 1.63 ± 0.15* — 0.75 0.24 0.76Metabolite 3.68 ± 0.26  5.89 ± 0.59** — 1.60 0.5  0.65

[0093] After iv administration of 100 mg/kg cordycepin, the nucleosidewas rapidly eliminated from mouse blood such that there was only a traceamount of cordycepin in serum samples collected at 5 min. In serumsamples collected 10 min later, cordycepin was not recovered. Neither3′-deoxyinosine nor cordycepin were detected in serum after oraladministration of 100 mg/kg cordycepin.

[0094] The most important finding of this study was made afteradministration of the azide derivative to mice. Following iv and oraladministration, a measurable amount of cordycepin was detected. Thepharmacokinetic profiles of cordycepin as well as the azide derivativeand the 3′-deoxyinosine metabolite are illustrated in FIG. 1.Pharmacokinetic parameters for the compounds are listed in Table 1.

[0095] The maximal measured concentrations (C_(max)) of the azidederivative were 143±33.4 μg/ml at 5 min after iv and 48.2±12.3 μg/ml at15 min. after oral administration. The half-life values were 0.35 h (iv)and 0.53 h (oral). Absolute bioavailability (F) of the azide derivativewas 0.94, but the difference between the AUC values for iv and oraladministration is statistically insignificant (Table 1).

[0096] The C_(max) values of cordycepin were 3.89±1.72 μg/ml at 5 min.after iv and 1.18±0.60 μg/ml at 30 minutes after oral administration.The half-life values were 0.24 h (iv) and 0.76 h (oral). The formulationwith the azide derivative of cordycepin allowed detection of cordycepinin serum above 0.05 μg/ml within 1.5 h (iv) and 3 h (oral). Althoughr_(e) for cordycepin was 0.75, there is no statistically significantdifference between the AUC values for iv and oral administration (Table1).

[0097] The AUC level of 3′-deoxyinosine was determined afteradministration of the azide derivative of cordycepin. The r_(e) was 1.6.The half-life of metabolite had the same range of value as the azidederivative and cordycepin.

[0098] These pharmacokinetic studies of the azide derivative ofcordycepin show that circulation of cordycepin in mouse serum issignificantly increased. The absolute bioavailability of the azidederivative after oral administration is 94% and amount of cordycepinreleased from the azide derivative is the same after iv and oraladministration. Oral administration of the azide derivative ofcordycepin results in a cordycepin concentration above the detectionlimit (0.05 μg/ml) for a longer period of time.

Example 3

[0099] Synthesis of6-azido-2′,3′-dideoxy-2′-fluoro-β-D-arabinofuranosylpurine (FAAddP) andN⁶-methyl-2′,3′-dideoxy-2′-fluoro-β-D-arabinofuranosyladenine (FMAddA),2′-F-ara-ddI azide prodrugs

[0100] Referring to Scheme 13, the prodrugs FAAddP (4) and FMAddA (5)were synthesized from the 6-chloropurine derivative 2. Compound 2 wassynthesized from5-O-benzoyl-3-deoxy-1,2-O-isopropylidine-α-D-ribofuranose (1) accordingto published procedures [Shanmuganathan, K. et al., “Enhanced braindelivery of an anti-HIV nucleoside 2′-F-ara-ddI by xanthine oxidasemediated biotransformation,” J. Med. Chem. (1994) 37:821-827]. Compound2 was debenzoylated using DIBAL-H in CH₂Cl₂ at −78° C. to obtaincompound 3. Upon treatment of compound 3 with LiN₃ in DMF at roomtemperature, the 6-azido derivative 4 was obtained in 73% yield. Thetreatment of compound 2 with methylamine in DMF at 80° C. for 5 hoursfollowed by the deprotection with saturated NH₃/MeOH for 15 hours gavecompound 5 quantitatively [Chu, C. K. et al., “Synthesis andstructure-activity relationships of 6-substituted 2′,3′-dideoxypurinenucleosides as potential anti-human immunodeficiency virus,” J. Med.Chem (1990) 33:1553-1561].

[0101] Melting points were determined on a Mel-Temp II laboratory deviceand are uncorrected. The ¹H NMR spectra were recorded on a JEOL FX 90 QFT spectrophotometer, with tetramethylsilane as the internal standard;chemical shifts are reported in parts per million (δ), and the signalsare quoted as s (singlet), d (doublet), t (triplet) m (multiplet), dm(double of multiplet) or brt (broad triplet). UV spectra were recordedon a Beckman DU-7 spectrophotometer. TLC were performed on Uniplates(silica gel) purchased from Analtech Co. Elemental analyses wereperformed by Atlantic Microlab, Inc., Norcross, Ga.

[0102]6Chloro-9-(5-O-benzoyl-2,3-dideoxy-2-fluoro-β-D-arabinofuranosyl)purine(2): Compound 2 was prepared from compound 1 according to previouslypublished procedures [Shanmuganathan, K. et al., “Enhanced braindelivery of an anti-HIV nucleoside 2′-F-ara-ddI by xanthine oxidasemediated biotransformation, J. Med. Chem. (1994) 37:821-827]. UV (MeOH)λ_(max) 263.5 nm (reported in Shanmuganathan, K. et al., “Enhanced braindelivery of an anti-HIV nucleoside 2′-F-ara-ddI by xanthine oxidasemediated biotransformation, J. Med. Chem. (1994) 37:821-827, as UV(MeOH) λ_(max) 263.5 nm).

[0103] 6-Chloro-9-(2,3-dideoxy-2-fluoro-β-D-arabinofuranosyl)purine (3):A solution of compound 2 (1.34 g, 3.56 mmol) in CH₂Cl₂ (40 mL) wascooled to −78° C. under nitrogen and DIBAL-H (10.5 mL, 1M solution inCH₂Cl₂) was added slowly. The reaction mixture was stirred at −78° C.for 45 min and quenched by the slow addition of MeOH. The reactionmixture was warmed to room temperature and the solvent was evaporated invacuo. The residue was dissolved in hot MeOH and filtered through a padof Celite. Upon concentration of the filtrate, the residue was purifiedon a silica gel column (5% MeOH in CHCl₃) to obtain pure compound 3(0.74 g, 75%): mp 157-158° C.; UV (MeOH) λ_(max) 263.5 nm (reported inBarchi, J. J. Jr. et al., “Potential anti-AIDS drugs. Lipophilic,adenosine deaminase-activated prodrugs,” J. Med. Chem. (1991)34:1647-1655 ,as UV (MeOH) λ_(max) 260 nm); [α]_(D) ²⁵ +52.3 (c 0.5,MeOH) (reported in Barchi et al., supra, as [α]_(D) ²⁵ +55.7 (c 1.4,MeOH)).

[0104] 6Azido-9-(2,3-dideoxy-2-fluoro-β-D-arabinofuranosylopurine (4): Asolution of 3 (1.0 g, 3.67 mmol) in DMF (25 mL) and LiN₃ (0.90 g, 18.3mmol) was stirred at room temperature for 24 hours. The DMF wasevaporated under high vacuum to yield a white solid which was boiled inMeOH and filtered 3 times to yield pure 4 (0.75 g, 73.5%) as a whitesolid: mp 209° C. (dec); UV (H₂O) λ_(max) 287.5 nm (ε7471, pH 7), 287.5nm (ε7327, pH 2), 234 nm (ε9478, pH 11); ¹H NMR (DMSO-d₆) δ2.03-2.95 (m,2H), 3.67 (brt, 1H), 5.09 (t, 1H, D₂O exchangeable), 5.57 (m, 1H), 6.61(dd, 1H), 8.84 (d, 1H), 10.15 (s, 1H); Anal. (C₁₀H₁₀FN₇O₂): C, H, N.

[0105] N⁶-Methyl-9-(2,3-dideoxy-2-fluoro-β-D-arabinofuranosyl)adenine(5)[See,Barchi, J. J. Jr. et al., “Potential anti-AIDS drugs. Lipophilic,adenosine deaminase-activated prodrugs,” J. Med. Chem. (1991)34:1647-1655; and Chu, C. K. et al., “Synthesis and structure-activityrelationships of 6-substituted 2′,3′-dideoxypurine nucleosides aspotential anti-human immunodeficiency virus,” J. Med. Chem. (1990)33:1553-1561]: A solution of 2 (1.60 g., 4.25 mmol) in DMF (50 mL) andmethylamine (3 mL) was sealed in a steel bomb and heated at 80° C. for 5hours. After cooling, the solvent was evaporated and NH₃ in MeOH (150mL) was added and stirred overnight. The evaporation of the solventyielded crude product which was purified by silica gel columnchromatography to yield pure 5 [See Barchi, J. J. Jr. et al. and Chu, C.K. et al, supra] (1.13 g, quantitative yield) as a hygroscopic foam: UV(MeOH) λ_(max) 264 nm (reported in Barchi et al. and Chu et al. as UV(MeOH) λ_(max) 265 nm); [α}_(D) ²⁵+56.1 (c 0.58, MeOH) (reported inBarchi et al., supra, as [α]_(D) ²⁵+56.57 (c 1.9, MeOH)).

[0106] In vitro stability in serum, brain and liver homogenate. Liverand brain homogenate were prepared in a 1: 1 (g:mL) ratio with isotonic0.05 M phosphate buffer, pH 7.4. FAAddP (70 μM) or FMAddA (50 μM) wereadded to the mouse serum, the brain homogenate or the liver homogenateand incubated in a shaker water bath at 37° C. Aliquots of 100 μL wereremoved at time zero and at selected times for up to 6 hours.Concentrations of the compounds were determined by HPLC.

[0107] Azido reduction assay. The analysis of azido reducing activitywas described previously [Cretton, E. M. and Sommadossi, J-P.,“Reduction of 2′-azido-2′,3′-dideoxynucleosides to their 3′-aminometabolite is mediated by cytochrome P-450 and NADPH-cytochrome P-450reductase in rat liver microsomes,” Drug Metab. Dispos. (1993)21:946-950].

[0108] Deamination of 2′-F-ara-ddA and FMAddA by adenosine deaminase.Samples (1.5 mL) of 2′-F-ara-ddA (80 μM) or FMAddA (50 μM) were preparedin 0.05 M isotonic phosphate buffer, pH 7.4, and placed into a shakingwater bath at 37° C. The reaction was initiated by the addition of 15 μLof adenosine deaminase (type VII from calf intestinal mucosa, SigmaChemical Co., St. Louis, Mo.). The final activity in the incubationmedia was 0.05 U/mL for 2′-F-ara-ddA and 1.0 U/mL for FMAddA. Atspecified time intervals, aliquots of 100 μL were withdrawn for thedetermination of 2′-F-ara-ddA and 2′-F-ara-ddI or FMAddA and2′-F-ara-ddI concentrations.

[0109] The biotransformation of the prodrug FAAddP to 2′-F-ara-ddIprobably involves a two-step metabolic process (Scheme 14). The prodrugwas first metabolized to 2′-F-ara-ddA by the P-450 reductase system. Asimilar reduction of the azido moiety of AZT to an amino function by thecytochrome P-450 system has been recently demonstrated [Placidi, L. etal., “Reduction of 3′-azido-3′-deoxythymidine to3′-amino-3′-deoxythymidine in human liver microsomes and itsrelationship to cytochrome P-450,” Clin. Pharmacol. Ther. (1993)54:168-176; Cretton, E. M. and Sommadossi, J-P., “Reduction of2′-azido-2′,3′-dideoxynucleosides to their 3′-amino metabolite ismediated by cytochrome P-450 and NADPH-cytochrome P-450 reductase in ratliver microsomes,” Drug Metab. Dispos (1993) 21:946-950]. 2′-F-ara-ddAwas then metabolized to 2′-F-ara-ddI by adenosine deaminase. The azidoreduction assay confirmed that FAAddP was metabolized to 2′-F-ara-ddAand 2′-F-ara-ddI by the microsomal fraction of the human liverhomogenate. Furthermore, in vitro biotransformation studies showed thatdirect conversion of FAAddP to 2′-F-ara-ddI by adenosine deaminaseoccurred at a negligible rate.

[0110] FAAddP was stable in phosphate buffer saline (PBS, pH 7.4) at 37°C. indicating that the compound is not susceptible to chemicalhydrolysis. The in vitro biotransformation of this prodrug in mouseserum, however, was relatively rapid with a degradation half-life of2.41 hours. Although FAAddP was metabolized in serum, no metaboliteswere identified. In the liver homogenate, FAAddP concentrations declinedin a biphasic fashion. Over the initial 45 minutes, the prodrug wasrapidly metabolized with a t_(½) of 0.48 hours. Subsequently, the rateof conversion was much slower (t_(½)=7.74 h) than the initial rateprobably due to the depletion of cofactors. The formation of2′-F-ara-ddI paralleled the decline of the prodrug with most of theprodrug being converted to 2′-F-ara-ddI. Only low concentrations of2′-F-ara-ddA were detected in the liver homogenate. Thebiotransformation of FAAddP in brain homogenate was somewhat slower thanthat in the liver with a half-life of 6.1 hours. However, only 10% ofthe prodrug was converted to 2′-F-ara-ddI and 5% to 2′-F-ara-ddA over a6 hour time period. Thus, similar to the studies in serum, anunidentified pathway was responsible for the disappearance of FAAddP inmouse brain.

[0111] The metabolic conversion of FMAddA to 2′-F-ara-ddI appeared to bea one-step process, facilitated by adenosine deaminase. The prodrug wasstable in PBS, mouse serum and mouse brain homogenate and was slowlymetabolized to 2′-F-ara-ddI in the liver homogenate (t_(½)=9.1 h).However, upon addition of adenosine deaminase to PBS (t_(½)=0.46 h) andbrain homogenate (t_(½)=3.7 h), virtually all of the prodrug wasconverted to 2′-F-ara-ddI.

[0112] Animal studies. The pharmacokinetics of the active nucleoside,2′-F-ara-ddI were investigated in mice. Animal studies were approved bythe University of Georgia Animal Care and Use Committee and conducted inaccordance with guidelines established by the Animal Welfare Act and theNational Institutes of Health Guide for the Care and Use of LaboratoryAnimals. Female NIH-Swiss mice (Harland Sprague-Dawley, Indianapolis,Ind.) weighing 24-28 g were housed in 12 h light/12 h darkconstant-temperature (22° C.) environment and had free access tostandard laboratory chow and water. Animals were acclimatized to thisenvironment for one week prior to the experiments.

[0113] 2′-F-ara-ddI, dissolved in physiological saline (15 mg/mL), wasadministered intravenously via tail vein injection at a dose of 20 mg/kg(79 μmoles/kg). FAAddP (55 mg/kg; 197 μmoles/kg) was administeredintravenously as a solution in DMSO (15 mg/mL) or orally by a gavage asa suspension in physiological saline. FMAddA, dissolved in saline (15mg/mL), was administered intravenously at a dose 112 mg/kg (437μmoles/kg). At selected time intervals, mice (three animals per eachtime point) were anesthetized with diethyl ether and sacrificed byexsanguination via left ventricular heart puncture. Serum was harvestedfrom blood collected. The brain was excised, rinsed with normal saline,blotted dry and weighed. Serum and brain samples were frozen at −20° C.until analysis.

[0114] Analytical Methodology. Concentrations of FAAddP, FMAddA,2′-F-ara-ddA and 2′-F-ara-ddI in PBS, serum, brain and liver homogenatewere determined by high performance liquid chromatography (HPLC). Thebrain or liver tissue were homogenized in a 1:1 (g:mL) ratio with icecold isotonic 0.05 M phosphate buffer, pH 7.4. Buffer, serum or tissuehomogenate (100 μL) was mixed with 10 μL of internal standard (25 μg/mLof 3′-azido-2′,3′-dideoxyuridine, AZddU). Acetonitrile (600 μL)containing 0.1% acetic acid was added while vortexing to precipitateproteins. The tubes were centrifuged at 3,000 rpm for five minutes andthe supernatant was transferred to a clean tube. Supernatant wasevaporated to dryness under a stream of nitrogen gas at roomtemperature. The residual film was reconstituted in 110 μL of mobilephase and 50 μL was injected onto the HPLC.

[0115] Chromatographic separations were performed using a Hypersil ODScolumn 150×4.5 mm, 5 μm particle size (Alltech Associates, Deerfield,Ill.) preceded by a guard column packed with 30-40 μm pellicularPerisorb RP-18. Mobile phase flow rate was 2 mL/min. For the analysis ofFAAddP and 2′-F-ara-ddA, the mobile phase consisted of 7% (v/v)acetonitrile in 80 mM sodium acetate, pH 5.0. The retention times for2′-F-ara-ddI, FAAddP and AZddU were 4.5, 7.9 and 5.1 min, respectively.The mobile phase for the analysis of 2′-F-ara-ddI consisted of 4.2%(v/v) acetonitrile in 40 mM sodium acetate, pH 4.1, yielding retentiontimes of 3.78 and 7.6 min, for 2′-F-ara-ddI and AZddU, respectively. Forthe analysis of FMAddA in serum and liver homogenate, a mobile phase of7.5% acetonitrile in 40 mM sodium acetate, pH 6.0 was used. Theretention time for FMAddA was 8.8 min and that for AZddU was 4.9 min.For FMAddA analysis in brain homogenate, the mobile phase consisted of7.5% acetonitrile in 10 mM K₂HPO₂ (pH 7.2) yielding retention times forFMAddA and AZddU of 7.8 and 4.3 min, respectively. Eluants weremonitored at a UV wavelength of 260 nm.

[0116] Nucleoside standards ranging from 0.04 μg/mL to 100 μg/mL,prepared in blank PBS, serum, brain homogenate and liver homogenate weretreated the same as unknown samples. Samples with nucleosideconcentrations greater than 100 μg/mL were diluted with the appropriateblank matrix. The limit of quantitation (signal-to-noise ratio of 3:1)for the 2′-fluoronucleosides in all biological media was 0.1 μg/mL.Extraction recovery was greater than 80% for all compounds. The intra-and inter assay relative standard deviations (RSDs) for each compoundwere less than 10% in all media.

[0117] Data analysis in vitro studies. Linear regression of the naturallogarithm of nucleoside analogue concentrations as a function of timewere used to determine first-order degradation rate constants (k) andassociated half-lives (t_(½)=0.693/k) in PBS, serum, liver homogenateand brain homogenate.

[0118] Data analysis in vivo studies. Nucleoside concentration as afunction of time data were analyzed by a non-compartmental technique.The AUC under the serum or brain nucleoside mean (n=3) concentrationversus time curve and the first moment (AUMC) were determined byLagrange polynomial interpolation and integration from time zero to thelast sample time (AUC_(0-τ)) with extrapolation to time infinity usingthe least-squares terminal slope (λ_(Z)) (Rocci, M. L. Jr. and Jusko, W.J., “LAGRAN program for ara and moments in pharmacokinetic analysis,”Comp. Prog. Biomed. (1983) 16:203-216]. The last 3 to 5 time points wereused to obtain λ_(Z). Half-life was calculated from 0.693/λ_(Z). Forintravenously administered compounds, total clearance (CL_(T)) wascalculated from Dose/AUC and steady-state volume of distribution(V_(SS)) from Dose×AUMC/AUC². The fraction of the prodrug converted toparent compound (f_(c)) was calculated from AUC_(←pd)×CL_(T)/Dose_(pd).where AUC_(p←pd) is the AUC of the parent compound after administrationof the prodrug (Dose_(pd)) and CL_(T) is the clearance of the parentcompound [Gibaldi, M. and Perrier, D., “Clearance concepts.” In:Pharmacokinetics, 2nd ed., Marcel Dekker Inc., New York (1982) 319-353].Relative brain exposure (r_(e)) was calculated fromAUC_(brain)/AUC_(serum).

[0119] Concentrations of 2′-F-ara-ddI in serum and brain afterintravenous administration of 20 mg/kg of the compound are illustratedin FIG. 2. Serum concentrations of 2′-F-ara-ddI declined rapidly with ahalf-life of 0.41 h (Table 2). Brain concentrations of the nucleosidepeaked at approximately 20 minutes, remained relatively constant for 30minutes, and subsequently declined in parallel with serumconcentrations. Relative brain exposure (r_(e)) of the2′-fluoronucleoside was 16.5 %. Total clearance of 2′-F-ara-ddI was 2.18L/h/kg and was moderate relative to the hepatic blood flow (5 L/h/kg)and renal blood flow (3.6 L/h/kg) in mice [Gerlowski, L. E. and Jain, R.K., “Physiologically based pharmacokinetic modeling: principles andapplications,” J. Pharm. Sci. (1983) 72:1103-1126]. Steady-state volumeof distribution was 0.78 L/kg hence, indicating that the compound wasdistributed intracellularly to a moderate extent. TABLE 2Pharmacokinetics parameters for 2′-F-ara-ddI, FAAddP, 2′-F-ara-ddA andFMAddA following administration of 20 mg/kg of 2′-F-ara-ddI or 55 mg/kgof FAAddP or 112 mg/kg of FMAddA to mice. Compound administered DoseRoute of AUC_(0-τ) AUG t_(½) AUC/Dose Compound measured (mg/kg)Administration Substrate (μM · h) (μM · h) (h) (μM · h/μmol/kg)2′-F-ara-ddI 2′-F-ara-ddI 20 iv Serum 36.2 38.0 0.41 0.48 Brain 5.6 6.30.47 0.08 FAAddP FAAddP 55 iv Serum 199.3 199.8 0.22 1.01 Brain 12.012.6 0.22 0.06 2′-F-ara-ddA Serum 3.0 8.6 2.92 0.04 Brain 3.9 4.8 0.910.02 2′-F-ara-ddI Serum 26.5 26.9 0.68 0.13 Brain 4.8 5.3 0.96 0.03FAAddP FAAddP 55 oral Serum 34.2 37.5 1.53 0.19 Brain  ND* ND ND ND2′-F-ara-ddA Serum 0.8 1.0 1.74 0.005 Brain ND ND ND ND 2′-F-ara-ddISerum 6.7 9.2 2.92 0.046 Brain ND ND ND ND FMAdda FMAdda 112  iv Serum296.8 298.8 0.46 0.68 Brain 39.0 39.5 0.98 0.09 2′-F-ara-ddI Serum 10.111.2 0.52 0.025 Brain ND ND ND ND

[0120] Concentrations of FAAddP, 2′-F-ara-ddA and 2′-F-ara-ddI in theserum and the brain after intravenous administration of 55 mg/kg ofFAAddP are shown in FIG. 3 (A and B). Serum concentrations of theprodrug declined rapidly with a half-life of 0.22 h (Table 2). Totalclearance and steady-state volume of distribution of the prodrug were1.12 L/h/kg and 0.58 L/kg, respectively. Thus, clearance of FAAddP wastwo-fold slower than that of 2′-F-ara-ddI and the distribution wasslightly less extensive. In serum samples, low levels of 2′-F-ara-ddAand higher concentrations of 2′-F-ara-ddI were observed. The eliminationhalf-life of 2′-F-ara-ddI after FAAddP administration was longer thanthat after the administration of 2′-F-ddI. The elimination half-life of2′-F-ara-ddA was 2.9 h. The higher concentration of 2′-F-ara-ddIcompared to 2′-F-ara-ddA is in agreement with the results of the invitro studies. Approximately 30% of the intravenously administered doseof the FAAddP was converted to 2′-F-ara-ddI.

[0121] FAAddP distributed rapidly into the brain with peak brain levelsobserved at the first sampling time (FIG. 2B). The relative brainexposure of the prodrug was 6.3 %, while that of 2′-F-ara-ddA and2′-F-ara-ddI were 55.8% and 19.7%, respectively. Thus, brain exposure toFAAddP was relatively low and the relative brain exposure to2′-F-ara-ddI after intravenous administration of FAAddP was similar tothat after administration of 2′-F-ara-ddI. Although the relative brainexposure for 2′-F-ara-ddA was relatively high, brain concentrations werelow.

[0122] To compare the disposition of 2′-F-ara-ddI after theadministration of FAAddP to that after the administration of2′-F-ara-ddI, AUC (area under curve) values were normalized for dose. Asshown in Table 2, the dose normalized AUC values for 2′-F-ara-ddI in theserum and the brain after intravenous administration of 55 mg/kg ofFAAddP were 3- to 4-fold lower than after the administration of 20 mg/kgof 2′-F-ara-ddI.

[0123] Concentrations of FAAddP, 2′-F-ara-ddA and 2′-F-ara-ddI in theserum after oral administration of 55 mg/kg FAAddP are depicted in FIG.3C. Absorption of FAAddP was rapid with peak serum concentrations of thecompounds achieved 0.5 hours after dosing. Oral bioavailability ofFAAddP was 19%, indicating incomplete absorption owing in part to itspoor solubility. Brain concentrations of FAAddP, 2′-F-ara-ddA and2′-F-ara-ddI were below the limit of quantitation because of the loworal bioavailability of FAAddP. Similar to intravenous study of FAAddP,higher concentrations of 2′-F-ara-ddI when compared to 2′-F-ara-ddA wereobserved, suggesting that the metabolism of FAAddP to 2′-F-ara-ddA isthe rate limiting step in the formation of 2′-F-ara-ddI.

[0124] As with FAAddP, a higher dose of FMAddA had to be administered tomeasure the levels of 2′-F-ara-ddI. Concentrations of FMAddA and2′-F-ara-ddI in serum and brain after intravenous administration of 112mg/kg of FMAddA are depicted in FIG. 4. Serum concentrations of FMAddAdeclined rapidly with a half-life of 0.45 h (Table 2). Total clearance(1.93 L/h/kg) and steady-state volume of distribution of FMAddA (0.79L/kg) were similar to those of 2′-F-ara-dedl. Only 5.6% of theadministered prodrug was converted to 2′-F-ara-ddI due to the low levelsof ADA in mice. The relative brain exposure of the prodrug was 7.5%;however, inconsistent with in vitro studies, no 2′-F-ara-ddI wasdetected in brain samples even at the relatively high dose administered.

[0125] In summary, FAAddP underwent reduction to 2′-F-ara-ddA followedby deamination to the active compound 2′-F-ara-ddI. FMAddA did notresult in increased brain delivery of the prodrug and was too slowlyconverted to 2′-F-ara-ddI to prove to be effective. In this study, a newapproach was demonstrated in the design of azido prodrugs by utilizingthe P-450 NADPh reductase system.

Example 5

[0126] Synthesis of 9-β-D-arabinofuranosyl-6-azidopurine (6-AAP) theazide derivative of AraA.

[0127] The azide derivative for Ara-A was synthesized according to thefollowing scheme 15:

[0128] The target compound 5 (6-AAP) was synthesized from ara-A (scheme15). Ara-A was deaminated to 9-(β-D-arabinofuranosyl)hypoxanthine (1)using adenosine deaminase in >90% yield. This method was found superiorin comparison to the deamination procedure with NaNO₂/AcOH. Compound Iwas peracetylated with acetic anhydride in pyridine, then converted toits 6-chloro derivative 3 under refluxing conditions with thionylchloride (26% from 1) [Robins, M. J. and Bason, G. L.,“6-Chloro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine from thechlorination of 2′-deoxyinosine,” In: Nucleic Acid Chemistry; Townsend,L. B., Tipson, R. S., Eds.; John-Wiley & Sons: New York (1978) Part II,pp. 601-606]. Compound 3 was deprotected to compound 4 by treatment withammonia in methanol and subsequently treated with LiN₃ in DMF to obtaincompound 5 (6-AAP) (38% from 3).

[0129] The stability of 6-AAP at pH 2, 7 and 11 and towards adenosinedeaminase hydrolysis was studied by UV spectroscopy. At pH 2 and 7,6-AAP did not show any significant change over a period of 2.75 hours at287.5 nm (37° C.). However, at pH 11, 6-AAP immediately changed its UVabsorption maximum from 287.5 nm to 222.5 nm. 6-AAP was not hydrolysedby adenosine deaminase for up to three hours in a separate in vitrostudy performed in phosphate buffer (pH 7.4) at 25° C. These resultsshow that unlike ara-A, 6-AAP is not a substrate for ADA.

[0130] Materials: Melting points were determined on a Mel-Temp IIlaboratory device and are uncorrected. The ¹H NMR spectra were recordedon a JEOL FX 90 Q FT spectrophotometer, with TMS as the internalstandard; chemical shifts are reported in parts per million (δ), and thesignals are quoted as s (singlet), d (doublet), t (triplet) or m(multiplet). UV spectra are recorded on a Beckman DU-650spectrophotometer. TLC was performed on Uniplates (silica gel) purchasedfrom Analtech Co. Elemental analysis was performed by Atlantic Microlab,Inc., Norcross, Ga. Ara-A and Adenosine deaminase (Type II crude powderfrom calf intestinal mucosa, 1-5 units/mg activity) was purchased fromSigma chemical Co., St. Louis, Mo. All other chemicals are of reagentgrade. Methanol and acetonitrile, methyl sulfoxide were purchased fromEM Science, Gibbstown, N.J.

[0131] 9-β-D-Arabinofuranosyl hypoxanthine (1): Method A. To a solutionof ara-A (500 mg, 1.87 mmol) in glacial acetic acid (8 ml), NaNO₂ (258mg, 3.73 mmol) dissolved in 1 ml of water, was added and stirred for sixhours. Then another three portions of NaNO₂ (200 mg, 2.8 mmol) each forevery six hours were added and stirring continued. After 36 hours, thesolvent was evaporated in vacuo and the residue was recrystallized fromhot water (25 ml) to obtain pure 1 (339 mg, 67.6%). UV(MeOH)λ_(max)249.0 nm, 207.0 nm.

[0132] Method B: To a suspension of ara-A (500 g, 1.87 mmol) indistilled water (30 ml) was added adenosine deaminase (4 mg), and themixture was stirred for 16 hours. Then the water was evaporated underreduced pressure and the white residue obtained was recrystallized fromhot water (20 ml) to obtain compound 1 as soft white solid (462 mg,92%). UV (MeOH) λ_(max) 248.5 nm, 205.5 nm.

[0133] 9-(2,3,5-Tri-O-acetylarabinofuranosyl)hypoxanthine (2): To asuspension of 1 (335 mg, 1.25 mmol) in dry pyridine (5 ml), aceticanhydride (1 ml 10.5 mmol) was added at 0° C. and the mixture wasstirred for 16 hours. Then the solvent was evaporated in vacuo, theresidue was dissolved in 50 ml of methylene chloride and was washed withwater (2×50 ml), sat. NaHCO₃ solution, brine and was dried (anhyd.sodium sulfate). The organic layer was concentrated in vacuo to obtain abrownish yellow solid 3 (339 mg, crude yield 68 %) which was used in thesubsequent reaction without any further purification. UV(MeOH) λ_(max)250.0 nm, 206.0 nm.

[0134] 6Chloro-9-(2,3,5-tri-Oacetyl-β-D-arabinofuranosyl)purine (3).Crude compound 2 (130 mg) was dissolved in dry CH₂Cl₂ (10 mL) and heatedto 55° C. Dry DMF (1 mL) followed by a 2 M solution of SOCl₂ in CH₃Cl₂(2.43 mL, 0.57 mmol) were added dropwise over a period of 45 minutes.The reaction mixture was gently refluxed for an additional 75 minutes.The reaction mixture was cooled to room temperature and diluted withCH₂Cl₂. The organic layer was washed with saturated NaHCO₃ solution(2×50 mL), brine (50 mL) and dried (anhydrous sodium sulfate). Theorganic phase was concentrated in vacuo and purified by preparative TLC(5 % MeOH/CHCl₃) to obtain pure 3 (50 mg, 26 % from 1): UV (MeOH)λ_(max)263.0 nm, 212.5 nm.

[0135] 9-(β-D-Arabinofuranosyl)-chloropurine (4). Compound3 (200 mg,0.5mmol) was dissolved in saturated NH₃/MeOH (5 mL) and stirred at roomtemperature for two hours. The solvent was evaporated in vacuo to obtaincrude 4 (170 mg) which was used for the subsequent reaction without anyfurther purification: UV (MeOH)λ_(max) 263.0 nm.

[0136] 9-(β-D-Arabinofuranosyl)-6azidopurine (5). A solution of 4 (170mg, 0.63 mmol) in DMF (5 ml) was treated with lithium azide (270 mg,5.52 mmol) and stirred for two days at room temperature. The solvent wasevaporated under reduced pressure at 40° C. and the crude oil wasrecrystallizd from MeOH to obtain pure 5 (67 mg, 38.4%): mp 185-190° C.(dec.); UVλ_(max) (water) pH 2: 205.0 (15,506), 287.0 (6,496); pH 7:208.5 nm (12,943), 287.5 nm (6033); pH 11: 222.5 nm (6,730); ¹H NMR(DMSO-d₆) δ3.66-3.90 (m, 3H, H-5′, H-4′), 4.16-4.33 (m, 2H, H-2′, H-3′),5.14 (t, 1H, 5′-OH, exchangeable with D₂O), 6.50 (d, 1H, H-1′), 8.75 (s,1H, H-8), 10.12 (s, 1H, H-2); IR (KBr) 2037, 1649; Anal.(C₁₀H₁₁N₇O₄.0.65 CH₃OH): C, H, N.

[0137] Stability studies of 6AAP. A kinetic study at varying pHs (pH 2,7 and 11 at 37.1° C.) was performed on a UV spectrophotometer toinvestigate the stability of 6-AAP. At pH 11, the UV absorption maximumfor compound 5 shifted from 287.5 nm to 222.5 nm immediately. At pH 7,the compound did not show any significant change in UV absorption maximaover a period of 2.75 hours at 287.5 nm indicating that it is stable atthe neutral pH. At pH 2, the compound was stable.

[0138] Comparative in vWtro studies were performed in mice liverhomogenate by separately incubating 6-AAP and 6-AAP/coformycin toinvestigate the biotransformation of 6-AAP alone and in the presence ofADA inhibitor, coformycin (Table 3). The half-lives of 6-AAP in theabsence and presence of coformycin were 4.90 hours and 5.98 hours,respectively. Ara-A was detected in both cases and the correspondinghalf-lives were 1.45 hours and 2.58 hours, respectively. When ara-Aalone was incubated in the mouse liver homogenate, its half-life was0.04 hours. Thus, the half-life of ara-A generated from 6-AAP alone was36 times greater than that of ara-A itself. Azido reduction assayutilizing the microsomal fraction of human liver homogenate alsoconfirmed that 6-AAP was converted to ara-A as shown in mice liverhomogenate studies. The biotransformation of 6-AAP to ara-A involves thecytochrome P450 NADPH dependent system (scheme 16). The stability andmetabolism of 6-AAP were also studied in mice serum and brain homogenate(Table 3). The half-lives of 6-AAP in mice serum and brain homogenatewere 3.73 hours and 7.29 hours, respectively. The decline of 6-AAP inserum was biphasic with a slow decline in the initial one hour period(T_(½)=3.73 h) and then with a faster decline rate (T_(½)=1.41 h). TABLE3 Parameters for the in vitro biotransformation of 6-AAP and ara-A inmice. K_(el) T_(1/2) Compound Medium Analyte (h⁻¹) (h) 6-AAP* Liverhomogenate 6-AAP 0.14 4.66 Ara-A 0.48 1.46 Ara-H 0.26 2.45 6-AAP andLiver homogenate coformycin 6-AAP 0.12 5.98 Ara-A 0.27 2.58 Ara-A Liverhomogenate Ara-A 16.87  0.04 Ara-H 0.31 2.24 6-AAP Serum^(§) 6-AAP 0.253.73 6-AAP Brain homogenate 6-AAP 0.01 7.29

[0139] Following these interesting in vitro results, in vivopharmacokinetic studies were performed in mice. FIG. 5 shows the meanserum concentrations of 6-AAP versus time after intravenous and oraladministration of 100 mg/kg of 6-AAP. The corresponding pharmacoldneticparameters for 6-AAP are presented in Table 4. Maximum concentration of6-AAP in serum was observed after five minutes of intravenous and after60 minutes of oral dosing (FIG. 5). Maximum concentrations of 6-AAP inserum after intravenous and oral dosing were 465±167 μg/mL and 7.8±2.51μg/mL, respectively. The terminal mean half-life values (0.55 hour and0.58 hour for intravenous and oral, respectively) were similar for bothroutes of administration. The area-under-curve (AUC) values for serumconcentration versus time for 6-AAP were 201.1±17.9 mg.h/L and 13.77±1.4mg.h/L, respectively following intravenous and oral administration of100 mg/kg of 6-AAP. After intravenous administration of 20 mg/kg of6-AAP, the AUC value was 85.6 mg.h/L (data not not shown), a 5-folddifference in the AUC values after intravenous dosing of 20 and toomg/kg of 6-AAP, which indicates that the disposition of 6-AAP in micefollowed linear kinetics in the dose interval between 20 mg/kg and 100mg/kg.

[0140] The concentrations of 6-AAP in brain versus time after itsintravenous and oral administration are shown in FIG. 5. Afterintravenous and oral administrations of 6-AAP, the maximumconcentrations of 5.92±0.7 and 1.87±μg/g in the brain were observed at 5and 30 minutes, respectively. The brain AUC values for 6-AAP were almostthe same for both (intravenous and oral) routes of administration(4.41±0.37 and 4.12±0.37 mg.h/L, respectively) (Table 4). The serum AUClevels of 6-AAP were 201±17.9 mg.h/L and 13.77±1.40 mg.h/L afterintravenous and oral administration of 6-AAP. In comparison, the brainAUC levels of 6-AAP were 2% and 33% of serum AUC levels afterintravenous and oral administration of 6-AAP, respectively. This datasuggests that there may be a saturable transport process of 6-AAP intothe brain. Half-life of 6-AAP in the brain was approximately two-foldgreater after oral administration (1.29 h) than that after intravenousadministration (0.77 h). The relative brain exposure (r_(e)) value of6-AAP was also greater after oral dosing (0.3) than that afterintravenous administration (0.02) of 6-AAP.

[0141] The serum concentrations of ara-A versus time after theadministration of ara-A (intravenous) and 6-AAP (oral and intravenous)are shown in FIG. 6. After the intravenous administration of ara-A, asignificant fraction of the compound was rapidly metabolized and itslevel declined from 18.5±3.5 μg/mL to 0.33±0.25 μg/mL in 25 minutes. TheAUC value was 3.95±0.2 mg.h/L and the half-life was 0.07 h. However, thepharmacokinetics curves for ara-A in serum after the intravenousadministration of 6-AAP were different from those after the intravenousadministration of ara-A. This curve revealed a “retard decline” of ara-Ain serum with a significant increase in the half-life (0.89 h). Ara-Alevel was 0.28±0.15 μg/mL after three hours of the injection. The AUCvalue (6.84±0.89 mg.h/mL) is 73% higher than that after the ara-Aadministration (3.95±0.20 mg.h/L) (Table 4). When 6-AAP (100 mg/kg) wasadministered orally, the serum AUC value of ara-A (1.15±0.13 mg.h/L) was29% of that after the intravenous administration of ara-A (3.95±0.20mg.h/L) and the half-life was 0.45 hours. TABLE 4 Pharmacokineticparameters of ara-A, 6-AAP and ara-A released from 6-AAP after dosing of100 mg/kg of ara-A or 6-AAP. Compound Route of Compound AUC t_(1/2)r_(e) Administered Administration Measured Tissue (mg.h/L) (h) (brain)ara-A iv ara-A serum 3.95 ± 0.20 0.07 — brain ND* — — 6-AAP iv 6-AAPserum 201.1 ± 17.9  0.55 — brain 4.41 ± 0.37 0.77 0.02 ara-A serum 6.84± 0.89 0.89 — brain 0.35 ± 0.04 1.47 0.05 6-AAP oral 6-AAP serum 13.77 ±1.40  0.58 — brain 4.12 ± 0.37 1.29 0.30 ara-A serum 1.15 ± 0.13 0.45 —brain 1.55 ± 0.57 5.03 1.35

[0142] The brain concentrations of ara-A after the administration of6-AAP versus time are illustrated in FIG. 7. Ara-A was not found in thebrain after its intravenous administration in a dose of 100 mg/kg.However, ara-A converted from 6-AAP exhibited a relatively constant meanconcentration in the brain of 0.3 to 0.1 ug/g from 5 minutes to 120minutes after intravenous administration and from 5 minutes to 240minutes after oral administration of 6-AAP. The greater distribution inthe brain was characterized by the increase in the brain AUC, r_(e) andhalf-life values for ara-A after oral administration (1.55±0.57 mg.h/L,1.35 and 5.03 hours, respectively) versus intravenous administration(0.35±0.04 mg.h/L, 0.05 and 1.47 hours, respectively) of 6-AAP (Table4).

[0143] Adenosine deaminase studies. 6-AAP (0.22 μM/mL) was incubatedwith adenosine deaminase (0.05 mg/mL) in phosphate buffer (pH 7.4) at25.1° C. and the change in the concentration was observed at 278.5 nmfor three hours.

[0144] Analysis. Concentrations of ara-A and 6-AAP in serum, brain(whole or homogenate) and liver homogenate were measured byhigh-performance liquid chromatography (HPLC). Chromatographicseparations were carried out on Millipore gradient system, which wasequipped with a Model 486 tunable UV detector, two Model 510 pumps, aModel 717 plus autosampler, and a Millennium 2010 Chromatography Managersoftware (Millipore Corporation, Milford, Mass.). All the solvents wereof HPLC grade. Chromatography was performed on an Altech Hypersil ODSC₁₈ (5 μm particle size, 4.5×150 mm, Altech Associates, Deerfield,Ill.). The mobile phase A was 0.5% acetonitrile in 2.5 mM KH₂PO₄(pH6.8), mobile phase B was 12% acetonitrile in 2.5 mM KH₂PO₄ (pH 6.8),mobile phase C was 2.5 mM KH₂PO₄ (pH 5.2) and mobile phase D was 23 %methanol in 2.5 mM KH₂PO₄.

[0145] In vitro metabolism study. Female NIH Swiss mice (HarlandSprague-Dawley, Indianapolis, Ind.) were sacrificed and serum, liver andbrain tissues were collected before each experiment. Serum was collectedfrom several animals. The brain and liver were washed in normal salineat 4° C., wiped and then weighed. Either 1 or 1.5 weight equivalents ofwater were added to the tissue and homogenized using a homogenizer. Thehomogenate was divided into two halves: one portion was used as theblank and the other for incubation with either ara-A, 6-AAP or&AAP/coformycin in a water bath shaker at 37° C. Initial concentrationsof 6-AAP and ara-A were 100 μg/mL. Samples and blanks of volume 400 μLwere collected at 0, 5, 15, and 30 minutes and at 1, 2, 3, 4 and 5hours.

[0146] To measure the analyte concentrations in serum, liver or brainhomogenate, 400 μL of sample was mixed with 50 μL of internal standard(AzdU, 5 μg/mL) and 0.7 mL of acetonitrile. After centrifugation, thesupernatant was decanted to another tube and treated with anhydrousNa₂SO₄, then vortexed for one minute and centrifuged again. The organiclayer was separated and evaporated under nitrogen stream at roomtemperature. The residue was reconstituted in mobile phase A andfiltered through MPS-I micropartition system (3KD membranes (AmiconInc., Beverly, Mass.) by centrifugation for 50 minutes at 2000 rpm tofurther clean-up the samples. 100 μL of filtrate was injected foranalysis.

[0147] For the HPLC analysis, during the first 10 minutes, the flow ratewas changed linearly from 1.5 mL/min to 1.0 mL/min and was continued at1.0 mL/min until the end of assay (67 minutes). In the first 20 minutes,the mobile phase consisted of 95% A and 5 % B; from 20 minutes, a lineargradient was run for 55 minutes to reach 5% A and 95% B. After eachanalysis, the column was equilibrated for 10 minutes to initialconditions. The λ_(max) was set at 249 nm for the first 15 minutes toobserve ara-H, from 15 to 35 minutes at 261 nm to observe ara-A, from 35to 45 minutes at 285 nm to observe 6-AAP, and subsequently changed to261 nm to observe AzdU. The retention time for ara-H, ara-A, 6-AAP andAzdU were 14.1, 33.8, 40.9 and 49.3 minutes, respectively.

[0148] Azido reduction study: The procedure for the analysis of azidoreducing activity was described previously [Cretton, E. M. andSommadossi, J.-P., “Reduction of 3′-azido-2′,3′-dideoxynucleosides totheir 3′-amino metabolite is mediated by cytochrome P-450 andNADPH-cytochrome P-450 reductase in rat liver microsomes,” Drug Metab.Dispos. (1993) 21:946-950]. Briefly, incubation mixtures containedeither 1.5 mg of human liver fraction protein (homogenate or supernatantfractions following centrifugation) or 1.5 mg microsomal protein, 5.0 mMMgCl₂, 6.0 mM NADPH and 0.4 mg/mL of 6-AAP in 0.1 M phosphate buffersaline at pH 7.4 (final volume of 0.2 mL). The reaction was initiated byadding NADPH and conducted at 37° C. for 60 minutes under nitrogen.Reactions were terminated by heating at 100° C. for 30 seconds and theproteins were removed by centrifugation at 14,000 g for six minutes.Aliquots (100 μL) were then analyzed for nucleosides by HPLC. Controlincubations were performed in the absence of protein.

[0149] Inhibition of azido reduction assay: Assays were performed using1.5 mg microsomal protein as described above following either a 45second exposure to carbon monoxide or a 5 minute pre-incubation with 1mM metyrapone prior to the addition of NADPH.

[0150] In vivo pharmacokinetics: Female NIH Swiss mice (HarlandSprague-Dawley, Indianapolis, Ind.) weighing 24-28 g were used for thepharmacokinetic experiments. Mice were acclimatized in a 12 h light/12 hdark, constant temperature (20° C.) environment for one week before theexperiments.

[0151] In a randomized study, animals were administered with either 20or 100 mg/kg of 6-AAP or 100 mg/kg of ara-A (intravenous). 6-AAP wasalso dosed orally (100 mg/kg p.o.). At least three animals each weresacrificed at 0.08, 0.025, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0hours after drug administration. Blood (serum) from the heart andwhole-brain samples were collected. Serum samples were treatedimmediately and brain samples were frozen at −20° C. until analysis. Todetermine the nucleoside concentrations in the serum, a know amount ofserum sample, 50 μL of the internal standard (AzdU, 5 μg/mL) and 1.0 mLof acetonitrile as a protein precipitant were added to polypropylenemicrocentrifuge tubes (1.7 mL). Tubes were mixed and centrifuged at9,000 rpm for 10 minutes.

[0152] To measure the ara-A and 6-AAP in the whole brain, 50 μL ofinternal standard (AzdU, 10 μg/ml) and 300 μL of water were added to theweighed tissue samples (approximately 300 mg). After homogenization, 1.8mL of acetonitrile was added to tissue homogenates, samples were mixedand centrifuged at 9,000 rpm for 10 minutes. The resulting supernatantfrom the serum or the brain was transferred to a clean tube and driedunder a stream of nitrogen gas at 22° C. The residue was reconstitutedin 220 μL of mobile phase D and after centrifugation at 12,000 rpm for40 min, 100-150 μL was injected for the HPLC analysis. During the first28 minutes, a linear gradient from 5% C and 95% D to 20% C and 80% D wasrun and then during the next 20 minutes, a linear gradient was run toreach 65% C and 35% D at a flow rate of 1.5 mL/min. After each assay,the column was equilibrated to initial conditions for 7 minutes. Theλ_(max) was set at 249 nm for the first 15 minutes to observe ara-H,from 15 to 30 minutes at 261 nm to observe ara-A, from 30 to 40 minutesat 285 nm to observe 6-AAP, and then changed to 261 nm to observe AzdU.The retention time for ara-H, ara-A, 6-AAP and AzdU were 13.5, 27.8,36.7 and 43.5 minutes, respectively.

[0153] Standard curves: Standard curves were prepared for each type ofsample by adding known amounts of ara-A and 6-AAP to the serum, brain orliver and subjecting them to the extraction procedure as describedabove. The limit of quantitation of the ara-A and 6-AAP were 0.1 μg/mLand 0.3 μg/mL, respectively. The percent recoveries of the compoundswere 63% for ara-A and 55% for 6-AAP.

[0154] Data analysis: Serum and tissue concentrations versus time datafor 6-AAP and ara-A were analyzed by noncompartmental methods. The areaunder concentration (AUC) versus time profiles from time zero to thelast measured concentration were determined by the linear trapezoidalrule and the AUC from the time of the last measured concentration toinfinity was determined by dividing the last determined concentration bythe least-squares elimination rate constant (λ_(Z)). Half-life wascalculated from 0.693/λ_(Z). The relative tissue exposure (r_(e)) of thecompounds was calculated from AUC_(Tissue)/AUC_(serum). A variation ofAUC was calculated according to previous procedures [Yuan, I.,“Estimation of variance for AUC in animal studies,” J. Pharm. Sci.(1993) 82:761-763].

[0155] 6-AAP kinetics in serum: Mean 6-AAP concentration versus timeprofiles for serum after intravenous and oral administration of 100mg/kg of 6-AAP were measured. The pharmacokinetic parameters for 6-AAPare presented in Table 4.

[0156] Maximum concentration of 6-AAP in serum was observed after 5 minof intravenous and after 60 min of oral dosing. Maximum concentrationsof 6-AAP in serum after iv and oral dosing were 465±167 μg/ml and7.8±2.51 μg/ml respectively. The terminal mean half-life values (0.55 hand 0.58 h for iv and oral respectively) were similar for both routes ofadministration. The AUC's for serum concentration versus time curve for6-AAP were 201.1±17.9 mg.h/L and 13.77±1.4 mg.h/L following intravenousand oral administration of 100 mg/kg. After intravenous administrationof 20 mg/kg of 6AAP, the AUC value was 85.6 mg.h/L. Thus, we found a5-fold difference in the AUC values after iv dosing of 20 and 100 mg/kgof 6-AAP. This indicates that the disposition of 6-AAP in mice followedlinear kinetics in the dose interval between 20 mg/kg and 100 mg/kg.Absolute bioavailability of 6-AAP was 6.8% following oraladministration.

[0157] While particular embodiments of the invention have been describedand exemplified, it will be understood that the invention is not limitedthereto, since many modifications can be made, and it is intended toinclude within the invention any such modifications as fall within thescope of the claims.

1. A pharmaceutical composition comprising: a) an azide derivative of adrug, which drug comprises an amino, carbonyl or hydroxy moiety, whereinin said azide derivative an azide group occurs at the site of saidamino, carbonyl or hydroxy moiety in place of said moiety, said azidederivative being capable of being converted to said drug in vivo byreplacement of said azide group with said amino, carbonyl or hydroxymoiety of said drug; b) a suitable pharmaceutical carrier. 2.N⁶-azido-β-D-3′-deoxyribofuranosyl purine, or a monophosphate,diphosphate or triphosphate or pharmaceutically acceptable salt thereof.3. 6-azido-2′,3′-dideoxy-2′-fluoro-β-D-arabinofurasylpurine or amonophosphate, diphosphate or triphosphate or pharmaceuticallyacceptable salt thereof.
 4. 9-(β-D-arabinofuranosyl)-6-azidopurine or amonophosphate, diphosphate or triphosphate or pharmaceuticallyacceptable salt thereof. 5.2-amino-6-azido-1,9-dihydro9[(2-hydroxyethoxy)methyl]-purineoramonophosphate,diphosphate or triphosphate or pharmaceutically acceptable salt thereof.6. 2-amino-6-azido-1,9-dihydro-9-[dihydroxymethyl]propyl-purine or amonophosphate, diphosphate or triphosphate or pharmaceuticallyacceptable salt thereof.
 7. The pharmaceutical composition of claim 1comprising an azide derivative selected from the group consisting ofazide derivatives of biologically active therapeutic purines andpyrimidines, nucleoside analogs and phosphorylated nucleoside analogs.8. The pharmaceutical composition of claim 1 comprising an azidederivative selected from the group consisting of azide derivatives ofaminoglycoside antibiotics.
 9. The pharmaceutical composition of claim 1comprising an azide derivative selected from the group consisting ofazide derivatives of ampicillin and ampicillin analogs.
 10. Thepharmaceutical composition of claim 1 comprising an azide derivativeselected from the group consisting of azide derivatives of sulfonamides.11. The pharmaceutical composition of claim 1 comprising an azidederivative selected from the group consisting of azide derivatives ofcephalosporin and cephalosporin analogs.
 12. The pharmaceuticalcomposition of claim 1 comprising an azide derivative of a biogeneticamine.
 13. The pharmaceutical composition of claim 1 comprising an azidederivative selected from the group consisting of azide derivatives ofalicyclic amines, ketones, or hydroxy-substituted compounds, includingaralkyl, heterocyclic aralkyl, and cyclic aliphatic compounds, where theamine or oxygen moiety is on the ring, or where the amine or oxygenmoiety is on an aliphatic side chain.
 14. A method of increasing thehalf-life of a drug in a subject, which drug comprises an amino,carbonyl or hydroxy moiety, comprising the steps of: (a) providing anazide derivative of said drug in which an azide group occurs at the siteof and in place of a carbonyl, hydroxy, or amine moiety of said drug,said azide derivative being capable of being reduced to the drug in thesubject's body by replacement of said azide group with said amino,carbonyl or hydroxy moiety; (b) administering said azide derivative to asubject.
 15. The method of claim 14 in which said drug is cordycepin.16. The method of claim 14 in which said drug is 2′-F-ara-ddI.
 17. Themethod of claim 14 in which said drug is AraA.
 18. The method of claim14 in which said drug is acyclovir.
 19. The method of claim 14 in whichsaid drug is penciclovir.
 20. The method of claim 14 in which said drugis selected from the group consisting of biologically active therapeuticalicyclic amines, ketones, and hydroxy-substituted compounds, includingaralkyl, heterocyclic aralkyl, and cyclic aliphatic compounds, where theamine or oxygen moiety is on the ring, or where the amine or oxygenmoiety is on an aliphatic side chain.
 21. The method of claim 14 inwhich said drug is selected from the group consisting of biologicallyactive therapeutic purines and pyrimidines, nucleoside analogs andphosphorylated nucleoside analogs.
 22. A method for ameliorating apathological condition in a patient, which pathological condition iscapable of being ameliorated by a selected drug which comprises anamino, carbonyl or hydroxy moiety, comprising treating the patient witha therapeutically effective azide compound which is capable ofmetabolizing in vivo to said selected drug by replacement of an azidegroup thereof with an amino, carbonyl or hydroxy moiety to form saiddrug effective for the treatment of said pathological condition.
 23. Themethod of claim 22 also comprising co-administering said azide compoundwith other therapeutic agents.